Substituted amino acids as erythropoietin mimetics

ABSTRACT

This invention relates to a series of substituted amino acids of Formula I                    
     pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention are small molecules which bind to the erythropoietin receptor and compete with the natural ligand for binding to this receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisonal of U.S. Ser. No. 09/517,926, filed Mar. 3, 2000, now U.S. Pat. No. 6,310,078, which is a continuation-in-part of U.S. Ser. No. 09/294,785, filed Apr. 19, 1999, now abandoned, which claimed benifit of U.S. Provisional 60/082,392, filed Apr. 20, 1998.

This invention relates to a series of small molecules which bind to the erythropoietin receptor and compete with the natural ligand for binding to said receptor. The invention includes pharmaceutical compositions containing these mimetics, their methods of production as well as intermediates used in their synthesis.

Erythropoietin (EPO) is a 34,000 dalton glycoprotein hormone which is produced in the mammalian kidney. Its primary role is stimulation of mitotic cell division and differentiation of erythrocyte precursor cells. As a result this hormone regulates the production of erythrocytes, the hemoglobin contained therein and the blood's ability to carry oxygen. The commercial product Epogen® is used in the treatment of anemia. This drug is produced by recombinant techniques and is formulated in aqueous isotonic sodium chloride/sodium citrate. Even though it has been used successfully in the treatment of anemia, it is a costly drug that is administered intravenously. This method of administration is both costly and inconvenient for the patient; therefore it would be desirable to find a EPO mimetic which has the potential for oral activity.

A small molecule EPO mimetic has advantages over the natural protein. The immune response associated with large peptides is unlikely to occur with small molecules. In addition, the variety of pharmaceutical formulations that may be used with small molecules are technically unfeasible for proteins. Thus the use of relatively inert formulations for small molecules is possible. The most important advantage of small molecules is their potential for oral activity. Such an agent would ease administration, cost less and facilitate patient compliance.

Although compounds which mimic EPO are useful in stimulating red blood cell synthesis, there are diseases where the overproduction of red blood cells is a problem. Erythroleukemia and polysythemia vera are examples of such diseases. Since EPO is an agent responsible for the maturation of red blood cell precursors, an antagonist of EPO would have utility treating either of those diseases.

SUMMARY OF THE INVENTION

The disclosed invention consists of a series of small molecules which demonstrate competitive binding with the natural ligand for the EPO receptor. As such these compounds are potentially useful in the treatment of diseases or conditions associated with this receptor. In addition, the invention contemplates methods of producing these compounds and intermediates used in their production.

The invention includes compounds of the Formula I:

wherein:

R¹ is the side chain of a natural or unnatural ax-amino acids, where if said side chain contains a protectable group, that group may be protected with a member of the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl, N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl, omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido, t-butyl, cinnamoyl, trityl, 4-methyltrityl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substituted phenylureido (where the phenyl substituents are phenoxy, halo, C₁₋₅alkoxycarbonyl);

R² and R³

may be taken together to form a six-membered aromatic ring which is fused to the depicted ring, or

are independently selected from the group consisting of hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy,

substituted phenyl (where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenylC₁₋₅alkyl (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenylC₁₋₅alkoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), and

substituted amino (where the substituents are selected from one or more members of the group consisting of C₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl, phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl, carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonyl substituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl, substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl, substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromatic phenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents are independently selected from one to five members of the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino]);

R⁴and R⁵

may be taken together to form a six-membered aromatic ring which is fused to the depicted ring, or

are independently selected from the group consisting of hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy,

substituted phenyl (where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenylC₁₋₅alkyl (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),

substituted phenylC₁₋₅alkoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), and

substituted amino (where the substituents are selected from one or more members of the group consisting of C₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl, phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl, carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonyl substituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl, substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl, substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromatic phenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents are independently selected from one to five members of the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino]);

W is selected from the group consisting of —CH═CH—, —S—, and —CH═N—;

Q is selected from the group consisting of —CH═CH—, —S—, and —CH═N—;

X is selected from the group consisting of carbonyl, C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5;

Y is selected from the group consisting of carbonyl, C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5;

n is 1, 2, or 3;

Z is selected from the group consisting of hydroxy, C₁₋₅ alkoxy, phenoxy, phenylC₁₋₅alkoxy, amino, C₁₋₅alkylamino, diC₁₋₅alkylamino, phenylamino, phenylC₁₋₅alkylamino, piperidin-1-yl

substituted piperidin-1-yl (where the substituents are selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, halo, aminocarbonyl, C₁₋₅alkoxycarbonyl, and oxo;

substituted phenylC₁₋₅alkylamino (where the aromatic substitutents are selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, phenylC₁₋₅alkenyloxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),

substituted phenoxy (where the aromatic substitutents are selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),

substituted phenylC₁₋₅alkoxy (where the aromatic substitutents are selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino), —OCH₂CH₂(OCH₂ ₂CH₂)_(s)OCH₂CH₂O—, —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—, —NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—, —NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—, —NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N, where s, p, and q are independently selected from 1-7

with the proviso that if n is 2, Z is not hydroxy, C₁₋₅ alkoxy, amino, C₁₋₅alkylamino, diC₁₋₅alkylamino, phenylamino, phenylC₁₋₅alkylamino, or piperidin-1-yl, with the further proviso that if n is 3, Z is (NH(CH₂)_(s))₃N.

and the salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The terms used in describing the invention are commonly used and known to those skilled in the art. “Independently” means that when there are more than one substituent, the substitutents may be different. The term “alkyl” refers to straight, cyclic and branched-chain alkyl groups and “alkoxy” refers O-alkyl where alkyl is as defined supra. “Cbz” refers to benzyloxycarbonyl. “Boc” refers to 1-butoxycarbonyl and “Ts” refers to toluenesulfonyl. “DCC” refers to 1,3-dicyclohexylcarbodiimide, “DMAP” refers to 4-N′,N-dimethylaminopyridine and “HOBT” refers to 1-hydroxybenzotriazole hydrate. “Fmoc” refers to N-(9-fluorenylmethoxycarbonyl), “DABCO” refers to 1,4-Diazabicyclo[2.2.2]octane, “EDCI” refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, “Dde” refers to 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, and “TMOF” refers to trimethyl orthoformate. The side chains of α-amino acids refer to the substituents of the stereogenic carbon of an α-amino acid. For example if the amino acid is lysine, the side chain is 1-aminobutan-4-yl. The term natural amino acid refers to the 20 α-amino acids of the L configuration which are found in natural proteins. Unnatural α-amino acids include synthetic amino acids such as, α-aminoadipic acid, 4-aminobutanoic acid, 6-aminohexanoic acid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine, α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, homocitrulline, homoserine, and statine as well as D-configuration amino acids. The term “protectable group” refers to a hydroxy, amino, carboxy, carboxamide, guanidine, amidine or a thiol groups on an amino acid side. Compounds of the invention may be prepared by following general procedures known to those skilled in the art, and those set forth herein.

The compounds of the invention may be prepared by liquid phase organic synthesis techniques or by using amino acids which are bound to a number of known resins. The underlying chemistry, namely, acylation and alkylation reactions, peptide protection and deprotection reactions as well as peptide coupling reactions use similar conditions and reagents. The main distinction between the two methods is in the starting materials. While the starting materials for the liquid phase syntheses are the N-protected amino acids or the lower alkyl ester derivatives of either the N-protected or N-unprotected amino acids, the starting material for the resin syntheses are N-protected amino acids which are bound to resins by their carboxy termini.

General Procedure for the Solid-Phase Synthesis of Symmetrical Nα,Nα-Disubstituted Amino Acids Scheme 1

An equivalent of an N-Fmoc-protected amino acid which is bound to a resin 1a is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. A solution of about two to three equivalents of an appropriately substituted halide, 1b, and a suitable base such DIEA is added to the resin bound amino acid and this mixture is shaken for 18-36 h. The resulting mixture is washed with several portions of a suitable solvent and is suspended and shaken in an acidic solution, such as 50% TFA/CH₂CH₂, over several hours to cleave the acid from the resin and give the N-disubstituted amino acid 1c.

By varying the resin bound amino acid 1a, one may obtain many of the compounds of the invention. The following resin bound amino acids may be used in Scheme I: alanine, N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine, β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester), S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid (β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine, leucine, N-ε-(2-chlorobenzyloxycarbonyl)lysine, N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline, O-(t-butyl)serine, O-(t-butyl)threonine, N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine, β-alanine, α-aminoadipic acid, 4-aminobutanoic acid, 6-aminohexanoic acid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine, α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, homocitrulline, homoserine, and statine. In addition, the choice of “W” and “X” can be varied by using known halide derivatives of 1b. For example using benzylchloride, 2-chloromethylthiophene, or 2-chloromethylpyridine gives compounds of the invention where “W” is —CH═CH—, —S—, or —CH═N—, respectively. For variations in “X”, the use of 2-chloroethylphenyl, 3-chloro-1-propenylbenzene, or benzeneacetyl chloride as 1b, give compounds where Y is (CH₂)₂, —CH═CH—CH₂—, or —CH₂C(O)— respectively. Still further, Scheme 1 may be used to produce combinatorial mixtures of products. Using mixtures of resin bound amino acids, 1a, with only one 1b produces said combinatorial mixtures. Alternatively, using one amino acid 1a with a mixture of 1b as well as mixture of 1a with mixtures of 1b gives a large range of combinatorial mixtures.

General Procedure for the Solid-Phase Synthesis of Unsymmetrical Nα,Nα-Disubstituted Amino Acids Scheme 2, Step A

An equivalent of an N-Fmoc-protected amino acid which is bound to a resin 1a is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. Trimethyl orthoformate and an appropriately substituted aldehyde 2a (5 equivalents) is added and the mixture is shaken under N₂ overnight. This mixture is treated with a suspension of NaBH(OAc)₃ (5 equivalents) in CH₂Cl₂ and shaken under N₂ overnight. After filtration and washing with a suitable solvent, the resulting product, resin bound Nα-monosubstituted amino acid 2b, is rinsed with a suitable solvent and its identity is confirmed by MS and or HPLC analysis after treatmet of a portion of the resin with 50% TFA/CH₂Cl₂.

Scheme 2, Step B

The resin 2b is suspended in an appropriate solvent such as DMF and is filtered. The appropriately substituted alkyl or arylkyl halide, 2c, and an appropriate base such as DIEA are added with some additional solvent and the mixture is shaken under N₂ for 18-36 h. The resin bound Nα,Nα-disubstituted amino acid, 2d, is isolated from the suspension and the resin is cleaved with an acidic solution to give the free acid 2e.

Scheme 3, Step C

A resin bound amine, 2d, where R⁴ is nitro, is suspended in a suitable solvent, such as DMF, and is filtered. This mixture is treated with SnCl₂ dihydrate in DMF and shaken under N₂ overnight. The solvent is removed and the resin is washed successive portions of a suitable solvent to give the resin bound compound 3a where R⁴ is amino. The resin is suspended in a suitable solvent and is combined with an organic base, such as pyridine an appropriately substituted carboxylic acid anhydride, acid chloride, or sulfonyl chloride. The mixture is shaken under N₂ overnight and is filtered to give the resin bound amino acid 3b. This material is treated with an acid and a suitable solvent to give the free amino acid 3b.

Scheme 3, Step D

The resin bound amine 3a is treated with TMOF and an appropriately substituted aldehyde 3c is added and the mixture is shaken under N₂ overnight. The resulting mixture is drained and treated with a suspension of NaBH(OAc)₃ in an appropriate solvent and this mixture is shaken under N₂ overnight. The resin bound 3-aralkylaminophenyl amino acid is identified my spectral techniques after clevage to give the free acid 3d as previously described.

Scheme 3, Step E

Resin bound, 2d, where R¹ is (CH₂)₄NH(Dde) is mixed with a suitable solvent, such as DMF, and shaken with successive portions of 2% solution of hydrazine hydrate in DMF over about 30 min. The resin is filtered and treated with a suitable solvent and a cyclic anhydride derivative 3e, and a base such as DMAP and pyridine. This mixture is shaken under N₂ overnight and filtered to give the resin bound amine, 3f. This material is identified by spectral techniques after clevage to give the free acid 3f as previously described.

Scheme 4, Step F

Resin bound 2b, where R² is nitro is suspended in CH₂Cl₂ and is treated with an organic base, such as pyridine, and 9-fluorenylmethoxy chloride. This mixture is shaken under N₂ overnight, filtered and resuspended in a suitable solvent. This mixture is treated with SnCl₂ dihydrate in DMF and shaken under N₂ overnight. The solvent is removed and the resin is washed successive portions of a suitable solvent and filtered to give the resin bound compound 4a where R² is amino. The resin 4a is then suspended in a suitable solvent, such as CH₂Cl₂, and is combined with 0.4 mmol of pyridine and 0.25-0.4 mmol of the appropriately substituted carboxylic acid anhydride, acid chloride, or sulfonyl chloride. The mixture is shaken under N₂ overnight, filtered, and washed successively with three portions each of CH₂Cl₂ and MeOH. This resin is suspended in DMF, filtered, and shaken under N₂ with 5 mL of a 40% solution of piperidine in DMF. After 1 h, the solvent is drained and the resin was washed successively with three portions each of suitable solvents to give the resin bound 4b. The identity of the compound was confirmed by spectral analysis after cleveage as previously described.

Scheme 5

The resin 2b (0.2 mmol) is suspended in CH₂Cl₂, filtered, and is resuspended in CH₂Cl₂. This suspension is treated with diethyl phosphonoacetic acid and diisopropylcarbodiimide or other suitable carbodiimide reagent, and the mixture is shaken under N₂ overnight. The solvent is drained and the resulting resin 5a was washed successively with three portions each of CH₂Cl₂ and MeOH. The resin is suspended in DMF and filtered. A solution of the appropriately substituted aldehyde 5b (0.6-1.0 mmol) in 3-5 mL of DMF, lithium bromide (0.6-1.0 mmol), and a suitable base such as DIEA or Et₃N (0.6-1.0 mmol) is added and the mixture is shaken under N₂ overnight. The solvent is removed and the resin is washed successively with three portions each of DMF, CH₂Cl₂, and MeOH. The identity of the resin bound substituted amino acid 5c was confirmed spectral techniques. The resin bound material may be treated with 50% TFA/CH₂Cl₂ over 1-1.5 h, to give the acid 5c.

Scheme 6

To prepare compounds where n is 2 and Z is NH(CH₂)_(s)NH, products of Schemes 1-5 may be used in Scheme 6. Treatment of two equivalents of the substituted amino acid 1c with an equivalent of the diamine 6a, in the presence of HOBT and a peptide coupling agent such as EDCI and a base such as DIEA at room temperature over 16 h gives the dimer 6b.

General Procedure for the Solution-Phase Synthesis of Symmetrical Nα,Nα-Disubstituted Amino Acids Scheme 7, Step A

A solution of of amino acid ester 7a, an appropriately substituted halide derivitive 1b, and an appropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in a suitable solvent, such as DMF, is heated at 50-100° C. under N₂ overnight, or until the starting material is exhausted, to give a mixture of the di and mono-substituted amines, 7b and 7c respectively. If the side chains of R¹ contain acid cleavable protecting groups, those groups may be cleaved by treatment with 30-80% TFA/CH₂Cl₂. Esters 7b and 7c may be independently converted to the corresponding acids 7d and 7e by hydrolysis with an appropriate base such as aqueous NaOH.

General Procedure for the Solution-Phase Synthesis of Unsymmetrical Nα,Nα-Disubstituted Amino Acids Scheme 8, Step A

A solution of 1 mmol of amino acid ester 8a (or the corresponding HCl salt and 1.1 mmol of DIEA) and 1-1.5 mmol of the appropriately substituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirred at room temperature under N₂ overnight. The solution was either concentrated and used directly for the next reaction, or was partitioned between EtOAc and water, washed with brine, dried over Na₂SO₄, and concentrated to give crude product, which was purified by MPLC to give mono-substituted product 8b.

Scheme 8, Step B

Amino ester 8b was dissolved in DMF, combined with 1.1-1.5 mmol of the appropriately substituted chloride or bromide 2c, and heated at 50-100° C. overnight. The reaction mixture was cooled and partitioned between water and EtOAc. The organic layer was washed three times with water and once with brine, dried over Na₂SO₄, and concentrated. The crude product was purified by MPLC to give pure 8c. For examples of 8c wherein the side chain R¹ contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 8c. For examples of 8c where R⁹ was equal to t-butyl, 8c was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h and treated as described above to give acid 8d. For examples of 8c where R⁹ was equal to methyl, ethyl, or other primary or secondary alkyl esters, 8c was stirred with with 1-2 mmol of aqueous LiOH, NaOH, or KOH in MeOH, EtOH, or THF at 20-80° C. until TLC indicated the absence of 8c. The solution was acidified to pH 4-5 with aqueous citric acid or HCl and was extracted with CH₂Cl₂ or EtOAc. The organic solution was washed with brine, dried over Na₂SO₄, and concentrated to give 8d.

Scheme 8, Step C

For examples of amino acid ester 8c where R¹=(CH₂)₄NHBoc, 8c (1 mmol) was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated to provide 8e as the TFA salt. Optionally, the TFA salt was dissolved in CH₂Cl₂ or EtOAc and washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, and concentrated to give 8e as the free base.

Scheme 8, Step D

A solution of 1 mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3e was stirred in CH₂Cl₂ or DMF under N₂ overnight. The resulting mixture was diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl, water, and brine, was dried over Na₂SO₄, and concentrated to provide 8f. Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted carboxylic acid anhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as above to provide 8g. Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCO was stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as above to provide 8h.

Scheme 9, Step A

For examples of 8c where R⁵=NO₂, a solution of 1 mmol of 8c (where R², R³, R⁴, or) and 10-12 mmol of SnCl₂ dihydrate was stirred in MeOH, EtOH, or DMF at 20-80° C. for 0.5-24 h under N₂. The solution was taken to room temperature and poured into aqueous Na₂CO₃ with rapid stirring. The resulting mixture was extracted with EtOAc or CH₂Cl₂ and the organic extracts were washed with brine, dried over Na₂SO₄, and concentrated to give the aminophenyl product 9a, which was purified by MPLC or used without further purification.

Scheme 9, Step B

A solution of 1 mmol of aminophenyl compound 9a and 1-1.5 mmol of the appropriately substituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirred at room temperature under N₂ overnight. The solution was either concentrated and used directly for the next reaction, or was partitioned between EtOAc and water, washed with brine, dried over Na₂SO₄, and concentrated to give crude product, which was purified by MPLC to give 9b. For examples of 9b wherein the side chain R¹ or R⁹ contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 9b was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 9b.

Scheme 9, Step C

A solution of 1 mmol of 3-aminophenyl compound 9a, 1.1-2 mmol of pyridine, and 1-1.5 mmol of the appropriately substituted acid chloride, acid anhydride, or sulfonyl chloride in 3-5 mL of CH₂Cl₂ or ClCH₂CH₂Cl was stirred at room temperature under N₂ overnight. The solution was partitioned between EtOAc and water, washed with water, saturated aqueous NaHCO₃, and brine, dried over Na₂SO₄, and concentrated to give crude product which was optionally purified by MPLC to give amide or sulfonamide 9c. For examples of 9c wherein the side chain R¹ or R⁹ contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 9c was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 9c.

General Procedure for the Solution-Phase Synthesis of Symmetrical Nα,Nα-Disubstituted Amino Amides and their Dimers and Trimers Scheme 10, Step A

A solution of 1 mmol of N-Cbz-protected amino acid 10a and the appropriate amine (ZH, 1 mmol), diamine (ZH₂, 0.5 mmol), or triamine (ZH₃ 0.33 mmol), was treated with 1.1 mmol of HOBt, 1.1 mmol of DIEA, and 2.1 mmol of EDCI in 3-6 mL of CH₂Cl₂ or DMF. [Alternatively, 1 mmol of the pentafluorophenyl ester or N-hydroxysuccinimide ester of 10a was mixed with the appropriate portion of amine (ZH), diamine (ZH₂), or triamine (ZH₃) in 3-6 mL of DMF.] The solution was stirred at room temperature under N₂ for 12-24 h, and EtOAc was added. The organic solution was washed with 5% aqueous citric acid, water, saturated NaHCO₃, and brine, dried over Na₂SO₄, and concentrated. The crude product was optionally purified by MPLC to afford amide 10b. Compound 10b was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated to provide the TFA salt which was dissolved in CH₂Cl₂ or EtOAc and washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, and concentrated to give 10c as the free base.

Scheme 10, Step B

A solution of 1 mmol of amino acid ester 10c (n=1), 2.5-3 mmol of the appropriately substituted chloride or bromide 2c, and 2.5-3 mmol of an appropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in 3-5 mL of DMF was heated at 50-100° C. under N₂ for 18-24 h. (For examples of 10c where n=2 or 3, the amounts of 2c and base were increased by two- or three-fold, respectively.) The reaction mixture was cooled and partitioned between water and EtOAc. The organic layer was washed three times with water and once with brine, dried over Na₂SO₄, and concentrated. The crude product was purified by MPLC to give pure amide 10d.

Alternatively, a solution of 1 mmol of amino acid ester 10c (n=1), 2.5-3 mmol of the appropriately substituted aldehyde 2a, and 2.5-3 mmol of borane-pyridine complex in 3-5 mL of DMF or EtOH was stirred at room temperature under N₂ for 3-5 days. (For examples of 10c where n=2 or 3, the amounts of 2c and borane-pyridine complex were increased by two- or three-fold, respectively.) The mixture was concentrated to dryness and was partitioned between water and CH₂Cl₂, washed with brine, dried over Na₂SO₄, and concentrated. The crude product was purified by MPLC to give pure amide 10d.

Scheme 10, Step C

For examples of 10d where R¹=CH₂CH₂CO₂-t-Bu or CH₂CO₂-t-Bu, 10d was stirred in 30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give acid 10e.

Scheme 10, Step D

For examples of 10d where R¹ is equal to (CH₂)₄NHBoc, 10d was stirred in 30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give amine 10f as the TFA salt which was optionally dissolved in CH₂Cl₂ or EtOAc, washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, and concentrated to give 10f as the free base.

Scheme 10, Step E

A solution of 1 mmol of 10f, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3e was stirred in CH₂Cl₂ or DMF under N₂ overnight. The resulting mixture was diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl, water, and brine, was dried over Na₂SO₄, and concentrated to provide acid 10g. Alternatively, 1 mmol of 10f, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted carboxylic acid anhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as above to provide 10h. Alternatively, 1 mmol of 8e, 14 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCO was stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as above to provide 10i.

General Procedure for the Solid-Phase Synthesis of Nα,Nα-Bis-Cinnamyl Amino Acids and Nα-Cinnamyl Amino Acids Scheme 11

An equivalent of an N-Fmoc-protected amino acid 11a which is bound to a polystyrene resin such as Wang resin is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. After filtration and washing with solvent, the resin is suspended in an appropriate solvent such as DMF. A solution of about 2-3 equivalents of an appropriately substituted halide 11b and a suitable base such DIEA is added to the resin bound amino acid and this mixture is shaken for 18-36 h. The resulting mixture is washed with several portions of a suitable solvent and is suspended and shaken in an acidic solution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acid from the resin to give a mixture of the Nα,Nα-bis-cinnamyl amino acid 11c and the Nα-cinnamyl amino acid 11d.

By varying the resin bound amino acid 11a, one may obtain many of the compounds of the invention. The following resin bound amino acids may be used in Scheme 11: alanine, N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine, β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester), S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid (β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine, leucine, N-ε-(2-chlorobenzyloxycarbonyl)lysine, N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline, O-(t-butyl)serine, O-(t-butyl)threonine, N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine, β-alanine, α-aminoadipic acid, 4-aminobutanoic acid, 6-aminohexanoic acid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine, α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, homocitrulline, homoserine, and statine.

Scheme 12, Step A

An equivalent of an N-Fmoc-protected amino acid which is bound to a resin 11a is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. After filtration and washing with solvent, the resin is suspended in an appropriate solvent such as trimethyl orthoformate (TMOF), an appropriately substituted aldehyde 12a (5 equivalents) is added, and the mixture is shaken under N₂ overnight. This mixture is treated with a suspension of NaBH(OAc)₃ (5 equivalents) in CH₂Cl₂ and shaken under N₂ overnight. After filtration and washing with a suitable solvent, the resulting product, resin bound Nα-monosubstituted amino acid 12b, is suspended and shaken in an acidic solution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acid from the resin to give the Nα-cinnamyl amino acid 11d.

Scheme 12, Step B

The resin 12b is suspended in an appropriate solvent such as DMF and is filtered. The appropriately substituted halide 12c and an appropriate base such as DIEA are added with some additional solvent and the mixture is shaken under N₂ for 18-36 h. The resin bound Nα,Nα-cinnamyl amino acid 12d is isolated from the suspension and the resin is cleaved with an acidic solution as described above to give the free acid 12e.

General Procedure for the Solution-Phase Synthesis of Nα,Nα-Bis-Cinnamyl Amino Acids and Nα-Cinnamyl Amino Acids Scheme 13

A solution of of amino acid ester 13a, an appropriately substituted halide 11b, and an appropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in a suitable solvent, such as DMF, is heated at 50-100° C. under N₂ overnight, or until the starting material is exhausted, to give a mixture of the Nα,Nα-bis-cinnamyl amino acid ester 13b and Nα-cinnamyl amino acid ester 13c. If the side chain of R¹ contains an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, those groups may be cleaved by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. For examples of 13b and 13c where the ester group R⁴ is a primary alkyl group such as methyl or ethyl, esters 13b and 13c may be independently converted to the corresponding acids 11c and 11d by hydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH. For examples of 13b and 13c where the ester group R⁴ is an acid-cleavable group such as t-butyl, esters 13b and 13c may be independently converted to the corresponding acids 11c and 11d by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Scheme 14, Step A

A solution of 1 mmol of amino acid ester and 1-1.5 mmol of the appropriately substituted aldehyde 12a in 3-5 mL of TMOF was stirred at room temperature under N₂ overnight. The solution was concentrated and used directly for the next reaction; optionally, the solution was partitioned between EtOAc and water, washed with brine, dried over Na₂SO₄, and concentrated to give crude product, which was purified by MPLC to give mono-substituted product 14a. For examples of 14a wherein the side chain R¹ contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was treated with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 14a. For examples of 14a where the ester group R⁴ is a primary alkyl group such as methyl or ethyl, esters 14a may be converted to the corresponding acids 11d by hydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH. For examples of 14a where the ester group R⁴ is an acid-cleavable group such as t-butyl, esters 14a may be converted to the corresponding acids 11d by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Scheme 14, Step B

Amino ester 14a was dissolved in DMF, combined with 1.1-1.5 mmol of the appropriately substituted chloride or bromide 12c, and heated at 50-100° C. overnight. The reaction mixture was cooled and partitioned between water and EtOAc. The organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated. The crude product was purified by MPLC to give pure 14b. For examples of 14b wherein the side chain R¹ contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was treated with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 14b. For examples of 14b where the ester group R⁴ is a primary alkyl group such as methyl or ethyl, esters 14b may be converted to the corresponding acids 12e by hydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH. For examples of 14b where the ester group R⁴ is an acid-cleavable group such as t-butyl, esters 14b may be converted to the corresponding acids 12e by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Although the claimed compounds are useful as competitive binders to the EPO receptor, some compounds are more active than others and are either preferred or particularly preferred.

The particularly preferred “R¹” s are the side chain of lysine, ornithine, arginine, aspartic acid, glutamic acid, glutamine, cysteine, methionine, serine, and threonine.

The particularly preferred “R² and R³” s are phenoxy, substituted phenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred “R⁴ and R⁵” s are phenoxy, substituted phenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred “W” is —CH═CH—

The particularly preferred “Q” is —CH═CH—

The particularly preferred “X” are C₁₋₅alkenyl and CH₂.

The particularly preferred “Y” are C₁₋₅alkenyl and CH₂.

The particularly preferred “n” are 1 and 2.

The particularly preferred “Z” are hydroxy, methoxy, phenethylamino, substituted phenethylamino, and —NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH—.

Pharmaceutically useful compositions the compounds of the present invention, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the compound of the present invention.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of EPO receptor-related activity is indicated. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, transdermal, oral and parenteral.

The term “chemical derivative” describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the EPO receptor or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitable topical, transdermal, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds according to this invention as the active ingredient for use in the modulation of EPO receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by transdermal delivery or injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, transdermal, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. The compounds of the present invention may be delivered by a wide variety of mechanisms, including but not limited to, transdermal delivery, or injection by needle or needle-less injection means. An effective but non-toxic amount of the compound desired can be employed as an EPO receptor modulating agent.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages of the EPO receptor modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds or modulators of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels, and other suitable polymers known to those skilled in the art.

For oral administration, the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed. The capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained. An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed. Alternatively the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal. Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried grains and the like. The active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.

The compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous, either by needle or needle-less means. The injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like. As an alternative, aqueous parenteral formulations may also be used. The vegetable oils are the preferred liquid carriers. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension. These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent. Formulations containing from 0.005 to 10% by weight of the active ingredient are acceptable. Preferred formulations are those containing from 0.01 to 5% by weight of the instant compounds or modulators.

The compounds of Formula I may be used in pharmaceutical compositions to treat patients (humans and other mammals) with disorders or conditions associated with the production of erythropoietin or modulated by the EPO receptor. The compounds can be administered in the manner of the commercially available product or by any oral or parenteral route (including but not limited to, intravenous, intraperitoneal, intramuscular, subcutaneous, dermal patch), where the preferred route is by injection. When the method of administration is intravenous infusion, compound of Formula I may be administered in a dose range of about 0.01 to 1 mg/kg/min. For oral administration, the dose range is about 0.1 to 100 mg/kg.

The pharmaceutical compositions can be prepared using conventional pharmaceutical excipients and compounding techniques. Oral dosage forms may be used and are elixirs, syrups, capsules, tablets and the like. Where the typical solid carrier is an inert substance such as lactose, starch, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like; and typical liquid oral excipients include ethanol, glycerol, water and the like. All excipients may be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known to those skilled in the art of preparing dosage forms. Parenteral dosage forms may be prepared using water or another sterile carrier.

Typically the compounds of Formula I are isolated as the free base, however when possible pharmaceutically acceptable salts can be prepared. Examples of such salts include hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic and saccharic.

In order to illustrate the invention the following examples are included. These examples do not limit the invention. They are only meant to suggest a method of practicing the invention. Those knowledgeable in chemical synthesis and the treatment of EPO related disorders may find other methods of practicing the invention. However those methods are deemed to be within the scope of this invention.

BIOLOGICAL EXAMPLES

The compounds of the invention were evaluated for the ability to compete with EPO in the following immobilized EPO receptor preparation (EBP-Ig, EPO binding protein-Ig).

EBP-Ig fusion protein (as disclosed in WO97/27219 which is herein incorporated by reference) was purified by affinity chromatography from the conditioned media of NSO cells engineered to express a recombinant gene construct which functionally joined the N-terminal 225 amino acids of the human EPO receptor and an Ig heavy chain as described herein. The interaction of biotin and streptavidin is frequently employed to capture and effectively immobilize reagents useful in assay protocols and has been employed here as a simple method to capture and immobilize EBP-Ig. EBP-Ig is initially randomly modified with an amine reactive derivative of biotin to produce biotinylated-EBP-Ig. Use of streptavidin coated plates allows the capture of the biotinylated EBP-Ig on the surface of a scintillant impregnated coated well (Flash plates, NEN-DuPont). Upon binding of [¹²⁵I]EPO to the ligand binding domain, specific distance requirements are satisfied and the scintillant is induced to emit light in response to the energy emitted by the radioligand. Unbound radioligand does not produce a measurable signal because the energy from the radioactive decay is too distant from the scintillant. The amount of light produced was quantified to estimate the amount of ligand binding. The specific assay format was suitable for the multi-well plate capacity of a Packard TopCount Microplate Scintillation counter. Compounds which were capable of reducing the amount of detected signal through competitive binding with the radioligand were identified.

Biotinylated EBP-Ig was prepared as follows. EBP-Ig (3 mL, OD₂₈₀ 2.9) was exchanged into 50 mM sodium bicarbonate, pH 8.5 using a Centriprep 10 ultrafiltration device. The final volume of the exchanged protein was 1.2 mL (OD₂₈₀ 2.6, representing about 2 mg total protein). 10 μL of a 4 mg/ml solution of NHS-LC-Biotin (Pierce) was added and the reaction mixture placed on ice in the dark for two hours. Unreacted biotin was removed by exchange of the reaction buffer into PBS in a Centriprep 10 device and the protein reagent aliquoted and stored at −70° C.

Each individual binding well (200 μL) contained final concentrations of 1 μg/mL of biotinylated EBP-Ig, 0.5 nM of [¹²⁵I]EPO (NEN Research Products, Boston, 100 μCi/μg) and 0-500 μM of test compound (from a 10-50 mM stock in 100% DMSO). All wells were adjusted to a final DMSO concentration of 5%. All assay points were performed in triplicate and with each experiment a standard curve for unlabelled EPO was performed at final concentration of 2000, 62, 15, 8, 4, and 0 nM. After all additions were made, the plate was covered with an adhesive top seal and placed in the dark at room temperature overnight. The next day all liquid was aspirated from the wells to limit analyte dependent quench of the signal, and the plates were counted on a Packard TOPCOUNT Microplate Scintillation Counter. Non-specific binding (NSB) was calculated as the mean CPM of the 2000 nM EPO wells and total binding (TB) as the mean of the wells with no added unlabelled EPO. Corrected total binding (CTB) was calculated as: TB−NSB=CTB. The concentration of test compound which reduced CTB to 50% was reported as the IC₅₀. Typically the IC₅₀ value for unlabelled EPO was ca. 2-7 nM and EMP1 was 0.1 μM. Table 1 lists the average % inhibition, and if determined the IC₅₀ and IC₃₀ values for compounds of Formula I, where the compound numbers refer to the compounds in the tables accompanying the preparative examples.

TABLE 1 Inhibition of EPO binding to EBP-Ig cpd % inh @ 50 μM IC₃₀, μM* IC₅₀, μM*  11 70 nd nd  12 59 nd nd  14 30 nd nd   15 48 nd nd   77 52 30 40   82 32 nd nd   86 37 nd nd 100 34 nd nd 101 32 nd nd 104 78 10 30 105 70 25 35 107 78 30 42 108 81 23 36 110 54 6 10 112 59 2 10 114 37 10 nd 115 35 nd nd 116 32 nd nd 117 34 nd nd 118 36 2 10 119 34 nd nd 120 35 nd nd 121 45 6 nd 137 60 5 30 139 46 2 10 178 36 nd nd 179 30 nd nd 183 36 nd nd 184 53 10 nd 203 37 50 nd 211 62 20 65 220 45 30 50 221 48 10 80 222 56 5 nd 224 51 25 50 227 48 20 50 230 42 nd nd 231 36 nd nd 235 49 20 50 237 55 30 70 238 39 nd nd 239 46 8 50 243 75 2 18 244 66 1 28 246 79 10 75 247 47 7 18 248 56 7 20 249 72 7 10 250 78 7 20 251 49 10 45 261 51 1.5 2 262 93 1 1.5 263 88 1 1.5 264 89 1.5 8 265 65 1 6 266 82 1 4 267 83 2 6 268 40 nd nd 269 55 8 85 270 56 7 100 271 77 2 7 272 78 5 10 285 41 nd nd 286 46 35 65 287 36 nd nd 300 57 35 145 305 48 35 225 312 45 10 85 321 42 45 nd 363 33 35 220 366 38 65 nd 368 40 90 nd *nd = not determined

PREPARATIVE EXAMPLES

Unless otherwise noted, materials used in the examples were obtained from commercial suppliers and were used without further purification. Melting points were determined on a Thomas Hoover apparatus and are uncorrected. Proton nuclear magnetic resonance (¹H NMR) spectra were measured in the indicated solvent with tetramethylsilane (TMS) as the internal standard using a Bruker AC-300 NMR spectrometer. NMR chemical shifts are expressed in parts per million (ppm) downfield from internal TMS using the d scale. ¹H NMR data are tabulated in order: multiplicity, (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), number of protons, coupling constant in Hertz). Electrospray (ES) mass spectra (MS) were determined on a Hewlett Packard Series 1090 LCMS Engine. Elemental analyses were performed by Quantitative Technologies, Inc. (QTI), PO Box 470, Salem Industrial Park, Bldg #5, Whitehouse, N.J. 08888-0470. Analytical thin layer chromatography (TLC) was done with Merck Silica Gel 60 F₂₅₄ plates (250 micron). Medium pressure liquid chromatography (MPLC) was done with Merck Silica Gel 60 (230-400 mesh).

Example 1 N,N-bis(3-Phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 96) and N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334)

A solution of 500 mg (1.97 mmol) of H-Glu(O-t-Bu)OMe.HCl, 997 mg (3.45 mmol) of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.; Kneen, G.; Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and 0.89 mL (5.1 mmol, 660 mg) of DIEA in 5 mL of DMF was stirred under N₂ at room temperature for 40 h. The mixture was partitioned between EtOAc and water and the organic layer was washed with water and brine. After drying over Na₂SO₄, the organic solution was concentrated to give 1.24 g of orange oil. The crude residue was purified by MPLC using a solvent gradient ranging from 10-30% EtOAc/hexanes to give two products. The less polar product (cpd 96, 235 mg, 19% based on starting amino acid), was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.39 (s, 9H), 2.0 (m, 2H), 2.33 (dt, 2H, J=2, 7 Hz), 3.24 (dd, 2H, J=8, 15 Hz), 3.5, (m, 3H), 3.69 (s, 3H), 6.13 (m, 2H), 6.47 (d, 2H, J=16 Hz), 6.86 (dd, 2H, J=1.5, 8 Hz), 7.0-7.4 (complex, 16H); MS (ES+) m/z 634 (MH+).

The more polar product (cpd 334, 422 mg, 50% based on starting amino acid) was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.42 (s, 9H), 1.9 (m, 2H), 2.35 (t, 2H, J=7.5 Hz), 3.2-3.4 (complex, 3H), 3.71 (s, 3H), 6.17 (dt, 1H), J=16, 6 Hz), 6.46 (d, 1H, J=16 Hz), 6.87 (dd, 1H, J=1.5, 8 Hz), 7.01 (m, 3H), 7.10 (t, 2H, J=7.5 Hz), 7.2-7.4 (complex, 3H); MS (ES+) m/z 426 (MH+). Anal. Calcd for C₂₅H₃₁NO₅: C, 70.57; H, 7.34; N, 3.29. Found: C, 70.29; H, 7.14; N, 3.08.

Example 2 N-(3-Phenoxycinnamyl)Glu-OMe (cpd 325)

A solution of 95 mg (0.22 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334) in 3 mL of 50% TFA/CH₂Cl₂ was stirred for 2 h at room temperature. The mixture was concentrated and the residue was dissolve in acetic acid and freeze-dried to give 117 mg of N-(3-phenoxycinnamyl)Glu-OMe (cpd 325)as an off-white solid; ¹H NMR (CD₃OD, 300 MHz) 2.3-2.7 (complex, 4H), 3.78 (s, 3H), 3.81 (d, 2H, J=7 Hz), 4.09 (t, 1H, J=5 Hz), 6.17 (dt, 1H, J=16, 7 Hz), 6.55 (d, 1H, J=16 Hz), 6.9 (m, 4H), 7.11 (t, 2H, J=7.5 Hz), 7.3 (m, 4H); MS (ES+) m/z 370 (MH+), 209. Anal. Calcd for C₂₁H₂₃NO₅.C₂HF₃O₂: C, 57.14; H, 5.00; N, 2.90. Found: C, 57.07; H, 5.02; N, 2.73.

Example 3 N,N-bis(3-Phenoxycinnamyl)Asp(O-t-Bu)-O-t-Bu (cpd 106)

A solution of 1.00 g (3.55 mmol) of Asp(O-t-Bu)-O-t-Bu.HCl, 2.05 g (7.1 mmol) of 3-phenoxycinnamyl bromide, and 1.86 mL (10.7 mmol, 1.38 g) of DIEA in 15 mL of DMF was heated under N₂ at 60° C. overnight. Additional 3-phenoxycinnamyl bromide (1.0 g, 3.4 mmol) and DIEA (0.95 mL, 0.705 g, 5.4 mmol) were added and heating was continued for an additional 14 h. The mixture was cooled and partitioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na₂SO₄. The solution was concentrated to give 3.5 g of an amber oil which was purified by MPLC using a solvent gradient ranging from 2.5-3% EtOAc/hexanes to afford 1.21 g of cpd 106 as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 9H), 1.48 (s, 9H), 2.49 (dd, 1H, J=7.5, 15.5 Hz), 2.70 (dd, 1H, J=7.5, 15.5 Hz), 3.26 (dd, 2H, J=7.5, 14.5 Hz), 3.47 (dd, 2H, J=4, 14.5 Hz), 3.88 (t, 1H, J=7.5), 6.13 (m, 2H), 6.48 (d, 2H, J=16 Hz), 6.86 (dd, 2H, J=2, 8 Hz), 7.0 (m, 6H), 7.1 (m, 4H), 7.2-7.4 (complex, 6H); MS (ES+) m/z 662 (MH+).

Example 4 N,N-bis(3-Phenoxycinnamyl)Asp-OH (cpd 107)

A solution of 1.14 g (1.62 mmol) of cpd 106 in 16 mL of 50% TFA/CH₂Cl₂ was stirred at room temperature for 24 h. The solution was concentrated and pumped to give 1.37 g (˜100%) cpd 107 as an amber oil; ¹H NMR (CD₃OD, 300 MHz) 3.1 (m, 2H), 4.0 (dd, 2H, J=8, 14 Hz), 4.27 (dd, 2H, J=8, 16 Hz), 4.70 (t, 1H, J=4 Hz), 6.38 (2H, dt, J=16, 8 Hz), 6.7-7.4 (complex, 20H); MS (ES−) m/z 562 ([M-H]+).

Example 5 N,N-bis(4-Benzyloxybenzyl)Lys(Boc)-OMe (cpd 111) and N-(4-Benzyloxybenzyl)Lys(Boc)-OMe

A solution of 594 mg (2.0 mmol) of Lys(Boc)-OMe.HCl, 524 mg (2.25 mmol) of 4-benzyloxybenzyl chloride, 75 mg (0.5 mmol), of NaI, and 0.61 mL (3.5 mol, 452 mg) of DIEA was warmed at 50-70° C. under N₂ overnight. The mixture was cooled and partioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na₂SO₄. The organic solution was concentrated to give 0.83 g of amber oil which was purified by MPLC using a solvent gradient ranging from 15-40% EtOAc/hexanes to give two products. The less polar product (296 mg), cpd 111, was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.28 (m, 4H), 1.43 (s, 9H), 1.70 (m, 2H), 3.03 (m, 2H), 3.28 (t, 1H, J=7 Hz), 3.40 (d, 2H, J=13.5 Hz), 3.74 (s, 3H), 3.81 (d, 2H, J=13.5 Hz), 5.05 (2, 4H), 6.92 (d, 4H, J=8.5), 7.23 (d, 4H, J=8.5), 7.25-7.5 (complex, 10H); MS (ES+) m/z 653 (MH+).

The more polar product (406 mg), N-(4-benzyloxybenzyl)Lys(Boc)-OMe, was isolated as a white solid; ¹H NMR (CDCl₃, 300 MHz) 1.4 (, 4H), 1.43 (s, 9H), 1.65 (m, 3H), 3.08 (m, 2H), 3.23 (t, 1H, J=6.5 Hz), 3.54 (d, 1H, J=12.5 Hz), 3.71 (s, 3H), 3.73 (d, 1H, J=12.5 Hz), 5.05 (s, 2H), 6.92 (d, 2H, J=8.5 Hz), 7.23 (d, 2H, J=8.5 Hz), 7.25-7.5 (complex, 5H); MS (ES+) m/z 457 (MH+).

Example 6 N-(4-Benzyloxybenzyl)-N-(3-nitrobenzyl)Lys(Boc)-OMe (cpd 113)

A solution of 374 mg (0.82 mmol) of N-(4-Benzyloxybenzyl)Lys(Boc)-OMe, 221 mg (1.03 mmol) of 4-nitrobenzyl bromide, and 197 L (1.13 mmol, 146 mg) of DIEA was warmed at 50-70° C. for 4 h, then at 40-50° C. overnight. After the addition of 0.2 mL of 1N aqueous HCl, the mixture was partioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na₂SO₄. The organic solution was concentrated to give 610 mg of an amber oil which was purified by MPLC 1:3 EtOAc/hexanes to afford 436 mg (90%) cpd 113 as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.35 (m, 4H), 1.42 (s, 9H), 1.75 (broad q, 2H, J=8 Hz), 3.06 (broad q, 2H, J=6 Hz), 3.28 (t, 1H, J=7.5 Hz), 3.48 (d, 1H, J=13.5 Hz), 3.66 (d, 1H, J=14.5 Hz), 3.76 (s, 3H), 3.79 (d, 1H, J=13.5 Hz), 3.97 (d, 1H, J=14.5 Hz), 4.47 (broad s, 1H), 5.05 (s, 2H), 6.93 (d, 2H, J=8.5 Hz), 7.22 (d, 2H, J=8.5 Hz), 7.3-7.5 (complex, 6H), 7.65 (d, 1H, J=7.5 Hz), 8.09 (d, 1H, J=8 Hz), 8.22 (s, 1H); MS (ES+) m/z 592 (MH+).

Example 7 N-(3-Aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe

A solution of 361 mg (0.61 mmol) of cpd 113 and 835 mg (3.7 mmol) of SnCl₂ dihydrate was stirred under N₂ at room temperature for 6 h. The slightly cloudy mixture was poured into 200 mL of 5% aqueous Na₂CO₃ with rapid stirring. The resulting milky suspension was extracted with three 75 mL portions of CH₂Cl₂ and the combined organic layers were washed with brine and dried over Na₂SO₄. The extracts were concentrated to give 344 mg of colorless oil which was purified by MPLC using 1:2 EtOAc/hexanes to provide 291 mg of N-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe as a yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.25 (m, 4H), 1.44 (s, 9H), 1.70 (m, 2H), 3.31 (dd, 1H, J=6, 9 Hz), 3.38 (d, 1H, J=14 Hz), 3.40 (d, 1H, J=13.5 Hz), 3.74 (s, 3H), 3.81 (d, 1H, J=14 Hz), 3.83 (d, 1H, J=13.5 Hz), 4.52 (broad s, 1H), 5.05 (s, 2H), 6.50 (broad d, 1H, J=8 Hz), 6.70 (m, 2H), 6.92 (d, 2H, J=8.5 Hz), 7.08 (t, 1H, J=7.5 Hz), 7.2-7.5 (complex, 7H); MS (ES+) m/z 562 (base, MH+), 506.

Example 8 N-(4-Benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys-OMe (cpd 117)

A solution of 42 mg (0.075 mmol) of N-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe and 12 μL (12 mg, 0.15 mmol) of pyridine in 0.5 mL of 1,2-dichloroethane was combined with 8.1 μL (11 mg, 0.083 mmol) and stirred under N₂ overnight. EtOAc (3 mL) was added and the solution was washed twice with 2 mL of water and 2 mL of saturated aqueous NaHCO₃. The EtOAc solution was filtered through a pad of Na₂SO₄ and concentrated to give 44 mg of N-(4-benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys(Boc)-OMe; MS (ES+) m/z 356 (MH+). The Boc-protected intermediate was stirred in 2 mL of 50% TFA/CH₂Cl₂ for 2 h and was concentrated and pumped at high vacuum to provide 66 mg of cpd 117 as the bis-TFA salt; ¹H NMR (CD₃OD, 300 MHz) 1.55 (m, 2H), 1.65 (m, 2H), 2.10 (m, 2H), 2.93 (t, 2H, J=7 Hz), 3.68 (t, 1H, J=7 Hz), 3.78 (s, 3H), 4.20 (m, 4H), 5.09 (s, 2H), 6.66 (dd, 1H, J=1.5, 3.5 Hz), 7.03 (d, 2H, J=8.5 Hz), 7.1-7.6 (complex, 11H), 7.76 (m, H), 8.07 (m, 1H); MS (ES+) m/z 556 (base, MH+), 360, 197.

Example 9 N,N-bis(3-Nitrobenzyl)Asp(O-t-Bu)-O-t-Bu (cpd 62)

A solution of 0.50 mg (1.77 mmol) of Asp(O-t-Bu)-O-t-Bu.HCl, 1.17 g (5.42 mmol) of 3-nitrobenzyl bromide, and 1.25 mL (0.93 g, 7.2 mmol) of DIEA in 6 mL of DMF was stirred at room temperature under N₂ for 24 h and was heated at 70-80° C. overnight. The reaction mixture was partitioned between EtOAc and water and the organic layer was washed twice with water and once with brine. After drying over Na₂SO₄, the organic solution was concentrated to give 0.86 g of a yellow oil which was purified by MPLC using 1:9 EtOAc/hexanes to afford 0.849 g (93%) cpd 62 as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.43 (s, 9H), 1.57 (s, 9H), 2.59 (dd, 1H, J=7.5, 16 Hz), 2.76 (dd, 1H, J=7, 16 Hz), 3.72 (t, 1H, J=7.5 Hz), 3.78 (d, 2H, J=14 Hz), 3.92 (d, 2H, J=14 Hz), 7.47 (t, 2H, J=8 Hz), 7.67 (d, 2H, J=7.5 Hz), 8.09 (broad d, 2H J=8 Hz), 8.16 (broad s, 2H); MS (ES+) m/z 538 (MNa+), 516 (base, MH+), 460, 404, 237.

Example 10 N,N-bis(3-Aminobenzyl)Asp(O-t-Bu)-O-t-Bu

A solution of 0.644 g (1.25 mmol) of cpd 62 and 2.82 g (12.5 mmol) of SnCl₂.2 H₂O in 12 mL of absolute EtOH was refluxed for 1.5 h. The mixture was cooled and poured into 300 mL of 5% aqueous Na₂CO₃ with rapid stirring. The cloudy mixture was extracted with three 150 mL portions of CH₂Cl₂ and the organic extracts were washed with brine and dried over Na₂SO₄. The CH₂Cl₂ solution was concentrated to afford 210 mg (37%) of N,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a cloudy yellow oil which was used without purification; ¹H NMR (CDCl₃, 300 MHz) 1.40 (s, 9H), 1.52 (s, 9H), 2.48 (dd, 1H, J=7, 16 Hz), 2.76 (dd, 1H, J=8 , 16 Hz), 3.48 (d, 2H, J=14 Hz), 3.55 (m, 1H), 3.73 (d, 2H, J=14 Hz), 6.56 (broad d, 2H J=8 Hz), 6.70 (broad s, 2H), 6.77 (d, 2H, J=7.5 Hz), 7.08 (t, 2H, J=8 Hz); MS (ES+) m/z 478 (MNa+), 456 (base, MH+), 400, 344.

Example 11 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu

To a solution of 109 mg (0.24 mmol) of N,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu, 29 mg (0.24 mmol) of DMAP, 125 μL (93 mg, 0.72 mmol) of DIEA in 1 mL of CH₂Cl₂ was added 95 μL (111 mg, 0.72 mmol) of 4-methylbenzoyl chloride. The solution was stirred under N₂ overnight and was then partitioned between EtOAc and water. The organic layer was washed with saturated aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated to give 177 mg of yellow oil. The crude material was purified by MPLC using a solvent gradient ranging from 20-25% EtOAc/hexanes to provide 87 mg of N,N-bis(3-(4-methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.36 (s, 9H), 1.55 (s, 9H), 2.35 (s, 6H), 2.53 (dd, 1H, J=6, 16 Hz), 2.76 (dd, 1H, J=9, 16 Hz), 3.69 (d, 2H, J=14), 3.77 (dd, 1H, J=6, 9 Hz), 3.83 (d, 2H, J=14), 7.01 (m, 6H), 7.26 (t, 2H, J=8 Hz), 7.59 (m, 6H), 8.11 (s, 2H), 8.49 (s, 2H); MS (ES+) m/z 714 (MNa+), 692 (base, MH+), 636, 580.

Example 12 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp-OH (cpd 64)

A solution of 87 mg (0.13 mmol) of N,N-bis(3-(4-methylbenzoyl)amino-benzyl)Asp(O-t-Bu)-O-t-Bu in 1 mL of 50% TFA/CH₂Cl₂ was stirred overnight. The mixture was concentrated and the residue was dissolved in HOAc and freeze-dried to afford 77 mg cpd 64 as a white solid; ¹H NMR (CD₃OD, 300 MHz) 2.40 (s, 6H), 2.85 (dd, 1H, J=6, 16.5 Hz), 2.98 (dd, 1H, J=8, 16.5 Hz), 4.02 (d, 2H, J=13.5 Hz), 4.08 (d, 4H, J=13.5 Hz), 4.10 (t, 1H, J=6.5 Hz), 7.22 (m, 6H), 7.34 (t, 2H, J=7.5 Hz), 7.60 (broad d, 2H, J=9 Hz), 7.76 (d, 4H, J=8 Hz), 7.88 (broad s, 2H); MS (ES+) m/z 580 (base, MH+).

Example 13 [N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

To a solution of 1.69 g (5.0 mmol) of N-Cbz-Glu(O-t-Bu)-OH, 0.365 mL (0.371 g, 2.5 mmol) of 1,8-diamino-3,6-dioxaoctane, 0.743 g (5.5 mmol) of HOBT, and 1.05 mL (0.776 g, 6.0 mmol) of DIEA in 15 mL of CH₂Cl₂ was added 1.05 g (5.5 mmol) of EDCI in one portion. After stirring at room temperature under N₂ overnight, the mixture was partitioned between EtOAc and 10% aqueous citric acid. The organic layer was washed with water, saturated NaHCO₃, and brine, dried over Na₂SO₄, and concentrated to give 1.87 g of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ as a colorless oil; ¹H NMR (CD₃OD, 300 MHz) 1.43 (s, 18H), 1.85 (m, 2H), 2.05 (m, 2H), 2.31 (t, 4H, J=8 Hz), 3.37 (t, 4H, J=5 Hz), 3.52 (t, 4H, J=5 Hz), 3.58 (s, 4H), 4.15 (m, 2H), 5.09 (dd, 4H, J=12, 16 Hz), 7.30 (m, 10H); MS (ES+) m/z 809 (base, MNa+), 787 (MH+).

Example 14 [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

Ammonium formate (0.78 g, 12.4 mmol) and 0.16 g of 10% palladium on carbon were added to a solution of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ in 12 mL of MeOH and the resulting suspension was stirred under N₂ at room temperature overnight. The mixture was diluted with CH₂Cl₂ and filtered through a Celite pad. The solids were washed thoroughly with CH₂Cl₂ and the combined organic filtrates were concentrated to dryness. The residue was partitioned between CH₂Cl₂ and saturated aqueous NaHCO₃, washed with brine, dried over Na₂SO₄, and concentrated to give 1.13 g of (Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ as a colorless oil; 1.44 (s, 18H), 1.81 (m, 2H), 2.08 (m, 2H), 2.35 (m, 4H), 3.39 (dd, 2H, J=5, 7.5 Hz), 3.47 (t, 4H, J=5 Hz), 3.58 (t, 4H, J=5 Hz), 7.53 (m, 2H).

Example 15 [N,N-bis(4-Benzyloxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (cpd 245)

A solution of 199 mg (0.384 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂, 403 mg (1.73 mmol) of 4-benzyloxybenzyl chloride, 30 mg (0.2 mmol) of NaI, and 334 L (248 mg, 1.92 mmol) of DIEA was stirred under N₂ at room temperature for several days. The solution was partitioned between EtOAc and water and the organic layer was washed three times with water and once with brine. After drying over Na₂SO₄, the solution was concentrated to give 528 mg of yellow oil which was purified by MPLC using a solvent gradient ranging from 42-50% EtOAc/hexanes to afford 318 mg (64%) of cpd 245 as a white foam; ¹H NMR (CDCl₃, 300 MHz) 1.42 (s, 18H), 2.01 (m, 4H), 2.38 (m, 2H), 2.55 (m, 2H), 3.03 (dd, 2H, J=5, 8 Hz), 3.31 (m, 2H), 3.4-3.6 (complex, 18H), 4.99 (s, 8H), 6.89 (d, 8H, J=8.5), 7.1-7.4 (complex, 30H).

Example 16 [N,N-bis(4-Benzyloxybenzyl)GluNHCH₂CH₂OCH₂]₂ (cpd 246)

A solution of 219 mg (0.168 mmol) of cpd 245 in 2 mL of 33% TFA/CH₂Cl₂ was stirred ad room temperature overnight. The mixture was concentrated to give a crude product which was dissolved in HOAc and freeze-dried to afford 251 mg of cpd 246 as an amber oil; ¹H NMR (CD₃OD, 300 MHz) 2.1-2.6 (complex, 8H), 3.3-3.6 (complex, 8H), 3.57 (s, 4H), 3.78 (m, 2H), 4.25 (broad d, 4H, J=14 Hz), 4.36 (broad d, 4H, J=14 Hz), 5.09 (s, 8H), 7.03 (d, 8H, J=8 Hz), 7.3-7.5 (complex, 28H); MS (ES+) m/z 1192 (MH+), 995, 596, 197 (base).

Example 17 [N-(3-Phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

A solution of 680 mg (0.76 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ and 278 μL (317 mg, 1.6 mmol) of 3-phenoxybenzaldehyde in 3 mL of TMOF was stirred overnight at room temperature under N₂. The mixture was concentrated and pumped at high vacuum to give a colorless oil which was dissolved in 3 mL of CH₂Cl₂ and treated with 678 mg (3.2 mmol) of NaBH(OAc)₃. After stirring under N₂ for 2 days, 50 mL of saturated aqueous NaHCO₃ was added and the mixture was extracted with CH₂Cl₂. The organic layers were combined, dried over Na₂SO₄, and concentrated and the crude product (1.01 g) was purified by MPLC using a solvent gradient ranging from 2-4% MeOH/CH₂Cl₂ to afford 490 mg of [N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as a colorless oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.89 (m, 4H), 2.31 (m, 4H), 3.12 (t, 2H, J=6 Hz), 3.45 (m, 8H), 3.55 (s, 4H), 3.60 (d, 2H, J=13.5 Hz), 3.73 (d, 2H, J=13.5 Hz), 6.86 (dd, 2H, J=1.5, 8 Hz), 7.00 (m, 8H), 7.2-7.4 (complex, 8H); MS (ES+) m/z 883 (MH+), 589, 442, 414, 386 (base), 183.

Example 18 [N-(3-Nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

DIEA (269 μL, 199 mg, 1.54 mmol), 3-nitrobenzyl bromide (322 mg, 1.49 mmol), and [N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (482 mg, 0.546 mmol) were combined in 2 mL of DMF and heated at 60-70° C. under N₂ for 2 days. The reaction mixture was cooled and partitioned between 100 mL of EtOAc and water. The organic layer was washed with three times with water and once with brine, dried over Na₂SO₄, and concentrated to give 661 mg (˜100%) of [N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ which was used without purification; MS (ES+) m/z 1154 (MH+), 577, 130 (base).

Example 19 [N-(3-Aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

A solution of 661 mg (0.54 mmol) of crude [N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ and 2.71 g (12.0 mmol) of SnCl₂•2 H₂O in 20 mL of absolute EtOH was refluxed under N₂ for 30 min. The cooled solution was poured into 500 mL of 2.5% aqueous Na₂CO₃ with rapid stirring and the resulting cloudy mixture was extracted thoroughly with EtOAc. The slightly cloudy organic extracts were washed twice with brine, dried over Na₂SO₄, anc concentrated to give 604 mg of yellow oil which was purified by MPLC using 3% MeOH/CH₂Cl₂ to provide 350 mg (59%) of [N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.97 (m, 4H), 2.25 (m, 4H), 2.48 (m, 4H), 3.03 (dd, 2H, J=5, 8 Hz), 3.30 (m, 2H), 3.4-3.8 (complex, 24H), 6.47 (d, 2H, J=7.5 Hz), 6.65 (m, 4H), 6.85 (d, 2H, J=9.5 Hz), 6.9-7.15 (complex, 12H), 7.2-7.4 (complex, 8H); MS (ES+) m/z 1094 (MH+), 547 (base).

Example 20 [N-(3-Phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu-NHCH₂CH₂OCH₂]₂ (cpd 247)

Pentanoyl chloride (16 uL, 16 mg, 0.136 mmol) was added dropwise to a solution of 68 mg (0.062 mmol) of [N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂, 20 μL (20 mg, 0.25 mmol) of pyridine in 0.3 mL of 1,2-dichloroethane. The mixture was shaken under N₂ overnight and was then partitioned between EtOAc and water. The organic layer was washed with saturated aqueous NaHCO₃, dried over Na₂SO₄, and concentrated to give 77 mg of [N-(3-phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂; MS (ES+) m/z 1073, 575 (base, MH+/2). The crude product was dissolved in 1 mL of 50% TFA/CH₂Cl₂ and allows to stand overnight. The solution was concentrated and the resulting oil was dissolved in HOAc and freeze-dried to provide 82 mg of cpd 247; ¹H NMR (CD₃OD, 300 MHz) 3.98 (t, 6H, J=7.5 Hz), 1.39 (sextet, 4H, J=7.5 Hz), 1.66 (quintet, 4H, J=7.5 Hz), 1.65 (m, 2H), 1.78 (m, 2H), 2.35 (t, 4H, J=7.5 Hz), 2.45 (m, 4H), 3.38 (m, 4H), 3.50 (t, 2H, J=5), 3.57 (m, 4H), 4.10 (broad s, 8H), 6.9-7.25 (complex, 14H), 7.25-7.4 (complex, 10H), 7.71 (s, 2H); MS (ES+) m/z 1150 (MH+), 575 (base).

Example 21 [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N

A solution of 1.0 g (2.63 mmol) of N-Cbz-Lys(Boc)OH, 0.131 mL (0.128 g, 0.876 mmol) of tris(2-aminoethyl)amine, 0.391 g (2.98 mmol) of HOBt, 0.555 g (2.89 mmol) of EDCI, and 0.55 mL (0.408 g, 3.16 mmol) of DIEA in 5 mL of CH₂Cl₂ was stirred under N₂ at room temperature overnight. The mixture was diluted with EtOAc and washed with 10% aqueous citric acid, saturated aqueous NaHCO₃, and brine. The solution was dried over Na₂SO₄ and concentrated to give 0.872 g of [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N as an off-white solid; ¹H NMR (CD₃OD, 300 MHz) 135 (m, 12H), 1.40 (s, 27H), 1.60 (m, 3H), 1.72 (m, 3H), 2.51 (m, 6H), 2.99 (m, 6H), 3.10 (m, 3H), 3.21 (m, 3H), 4.12 (m, 3H), 5.00 (d, 3H, J=12.5 Hz), 5.08 (d, 3H, J=12.5 Hz), 7.29 (m, 15H); MS (ES+) m/z 1243 (base, MH+), 567, 467.

Example 22 [Lys(Boc)-NHCH₂CH₂]₃N

A solution of 0.841 g (0.682 mmol) [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N, 0.252 g of 10% Pd—C, and 0.774 g (12.3 mmol) of ammonium formate in 10 mL of MeOH was stirred for 5 h at room temperature under N₂. The mixture was filtered through a Celite pad, the solids were washed with CH₂Cl₂, and the reslulting solution was concentrated to dryness. The residue was partitioned between CH₂Cl₂ and brine; the organic layer was dried over Na₂SO₄ and concentrated to provide 0.191 g of [Lys(Boc)-NHCH₂CH₂]₃N as an off-white solid; ¹H NMR (CD₃OD, 300 MHz) 1.40 (s, 27H), 1.45 (m, 12H), 1.75 (m, 6H), 2.62 (m, 6H), 3.01 (m, 6H), 3.28 (m, 6H), 3.64 (m, 3H); MS (ES+) m/z 853 (MNa+), 831 (MH+), 266 (base).

Example 23 [N,N-bis(3-Phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N

A solution of 65 mg (0.078 mmol) of [Lys(Boc)-NHCH₂CH₂]₃N, 120 μL (140 mg, 0.70 mmol) of 3-phenoxybenzaldehyde, and 71 μL (65 mg, 0.70 mmol) of borane-pyridine complexin 3 mL of absolute EtOH was stirred for 4 days at room temperature under N₂. The mixture was concentrated to dryness and partitioned between water and CH₂Cl₂. The organic layer was concentrated to give a yellow oil which was purified by MPLC using 2.5% MeOH/CH₂Cl₂ to give 78 mg of [N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N as a yellow oil; MS (ES+) m/z 872 (base, [M-C₁₃H₁₂O)/2]+), 611, 443.

Example 24 [N,N-bis(3-Phenoxybenzyl)Lys-NHCH₂CH₂]₃N (cpd 277)

A solution of 78 mg (0.048 mmol) of [N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N in 2 mL of 50% TFA/CH₂Cl₂ was stirred for 2 h at room temperature. The mixture was diluted with CH₂Cl₂, washed with water and 5% Na₂CO₃, and concentrated to give 57 mg of cpd 277 as an off-white foam; ¹H NMR (CD₃OD, 300 MHz) 1.35 (m, 6H), 1.52 (m, 6H), 1.76 (m, 6H), 2.75 (m, 6H), 3.19 (m, 6H), 3.40 (m, 6H), 3.60 (m, 9H), 3.77 (m, 6H), 6.79 (d, 6H, J=8 Hz), 6.93 (m, 24H), 7.05 (m, 6H), 7.19 (m, 6H), 7.29 (m, 12H); MS (ES+) m/z 813 ([MH₂/2]+), 721, 542 (base, [MH/3]+).

Example 25 N,N-bis(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290) and N-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352)

A solution of 423 mg (2.0 mmol) of H-Ser(t-Bu)OMe.HCl, 1.01 g (3.5 mmol) of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.; Kneen, G.; Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and 0.87 mL (5.0 mmol, 650 mg) of DIEA in 6 mL of DMF was stirred under N₂ at room temperature for 20 h. The mixture was partitioned between EtOAc and water and the organic layer was washed with water and brine. After drying over Na₂SO₄, the organic solution was concentrated to give 0.98 g of yellow oil. The crude residue was purified by MPLC using a solvent gradient ranging from 10-30% EtOAc/hexanes to give two products. The less polar product (168 mg, 14% based on starting amino acid), N,N-bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290), was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 3.35 (dd, 2H, J=7, 14.5 Hz), 3.53 (dd, 2H, J=5.5, 14.5 Hz), 3.6-3.8 (complex, 3H), 3.69 (s, 3H), 6.18 (dt, 2H, J=16, 6.5 Hz), 6.49 (d, 2H, J=16 Hz), 6.86 (dd, 2H, J=2, 8 Hz), 6.9-7.4 (complex, 16H); MS (ES+) m/z 614, 592 (MH+, base), 406, 384, 209.

The more polar product (354 mg, 46% based on starting amino acid), N-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352), was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 1.98 (broad s, 1H), 3.32 (ddd, 1H, J=1.2, 6.5, 14 Hz), 3.4-3.7 (complex, 4H), 3.72 (s, 3H), 6.21 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=16 Hz), 6.88 (dd, 1H, J=1.5, 8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 789 (2M+Na+), 384 (MH+, base), 209.

Example 26 N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299)

N,N-Bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290, 168 mg, 0.284 mmol) was stirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. The solvent was removed using a rotary evaporator and the crude residue was partitioned between EtOAc and saturated aqueous NaHCO₃. After washing with brine and drying over Na₂SO₄, the organic layer was concentrated using a rotary evaporator and the crude product (134 mg) was purified by MPLC using 30% EtOAc/hexanes to give 44 mg (29%) of N,N-bis(3-phenoxycinnamyl)Ser-OMe (cpd 299) as a colorless oil; ¹H NMR (CDCl₃, 300 MHz) 1.6 (broad s, 2H), 3.38 (dd, 2H, J=8, 12 Hz), 3.4-3.9 (complex, 5H), 3.72 (s, 3H), 6.13 (dt, 2H, J=16, 7 Hz), 6.50 (d, 2H, J=16 Hz), 6.8-7.4 (complex, 18H); MS (ES+) m/z 536 (MH+).

Example 27 N,N-Bis(3-phenoxycinnamyl)Ser-OH (cpd 300)

N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299, 44 mg, 0.082 mmol) was dissolved in 0.2 mL of MeOH and was stirred with 0.090 mL of 1N aqueous NaOH. When TLC analysis revealed that starting material had been consumed, the solvent was removed by rotary evaporation and the residue was lyophilized from acetic acid to give 42 mg (88%) of N,N-bis(3-phenoxycinnamyl)Ser-OH acetate (cpd 300) as a sticky yellow solid; ¹H NMR (methanol-d₄, 300 MHz) 1.97 (s, 3H), 3.3-4.2 (complex, 7H), 6.80 (d, 2H, J=16 Hz), 6.9-7.4 (complex, 18H); MS (ES+) m/z 522 (MH+), 209.

Example 28 N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346)

N-(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352, 268 mg, 0.699 mmol) was stirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. The solvent was removed using a rotary evaporator and the crude residue (256 mg) was purified by MPLC using EtOAc to give 137 mg (60%) of N-(3-phenoxycinnamyl)Ser-OMe (cpd 346) as a colorless oil; ¹H NMR (CDCl₃, 300 MHz) 2.2 (broad s, 2H), 3.36 (dd, 1H, J=6, 14 Hz), 3.4-3.5 (complex, 2H), 3.62 (dd, 1H, J=6.5, 11 Hz), 3.74 (s, 3H), 3.80 (dd, 1H, J=4.5, 11 Hz). 6.19 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=6 Hz), 6.88 (dd, 1H, J=1.5, 8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 677 (2M+Na+), 350 (M+Na+), 328 (MH+), 209 (base).

Example 29 N-(3-phenoxycinnamyl)Ser-OH (cpd 347)

N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346, 110 mg, 0.336 mmol) was dissolved in 1.5 mL of MeOH and was stirred with 0.50 mL of 1N aqueous NaOH. When TLC analysis revealed that starting material had been consumed, the solvent was removed by rotary evaporation. The residue was dissolved in water and acidified to pH 7-8 with 1N aqueous HCl; the resulting solids were filtered, washed with water, and dried to give 71 mg of white powder. The insoluble powder was dissolved in TFA and, after removal of excess TFA by rotary evaporation, lyophilized from acetic acid to give 82 mg (57%) of N-(3-phenoxycinnamyl)Ser-OH trifluoroacetate (cpd 347) as an amber oil; ¹H NMR (methanol-d₄, 300 MHz) 3.88 (d, 2H, J=7H), 4.0-4.2 (complex, 3H), 6.27 (dt, 1H, J=16, 6.5), 6.83 (d, 1H, J=16 Hz), 6.9-7.4 (complex, 9H); MS (ES+) m/z 314, (MH+), 209.

Example 30 N-(3-Phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337)

A mixture of 249 mg (0.585 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334) in 3 mL of MeOH was sonicated to speed dissolution, and the resulting solution was treated with 0.585 mL of 1N aqueous NaOH. After stirring overnight, the MeOH was removed using a rotary evaporator and the residue was dissolved in water. Acidification with 0.64 mL of 1N aqueous HCl produced a 250 mg of solid material that was triturated with Et₂O to give 111 mg (46%) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337) as a white solid; ¹H NMR (300 MHz, methanol-d₄) 1.43 (s, 9H), 1.9-2.2 (complex, 2H), 2.46 (t, 2H, J=7 Hz), 3.57 (dd, 1H, J=5, 7 Hz), 3.78 (dd, 1H, J=7, 13.5 Hz), 3.82 (dd, 1H, J=7, 13.5 Hz), 6.28 (dt, 1H, J=16, 7 Hz), 6.81 (d, 1H, J=16 Hz), 6.9-7.5 (complex, 9H); MS (ES+) m/z 412 (MH+, base), 356, 209. Anal. Calcd for C₂₄H₂₉NO₅.0.4 H2O: C, 68.55; H, 7.04; N, 3.24. Found: C, 68.89; H, 7.04; N, 3.24.

Example 31 N-(3-Phenoxycinnamyl)Glu-OH (cpd 326)

A mixture of 85 mg (0.21 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337) in was stirred in 1 mL of 50% TFA/CH₂Cl₂ for 1 h. After solvent removal using a rotary evaporator, the residue was dissolved in acetic acid and freeze-dried to give 75 mg (76%) of N-(3-phenoxycinnamyl)Glu-OH tifluoroacetate (cpd 326) as a fluffy white solid; ¹H NMR (300 MHz, methanol-d₄) 2.0-2.4 (complex, 2H), 2.55 (m, 2H), 3.84 (d, 2H, J=7 Hz), 3.96 (dd, 1H, J=5, 7 Hz, 6.24 (dt, 1H, J=16, 7 Hz), 6.84 (d, 1H, J=16 Hz), 6.9-7.4 (complex, 9H); MS (ES+) m/z 356 (MH+), 209 (base).

TABLE 2

R¹ (amino cpd % inh acid side chain) R² R³ W, Q 11 70 Asn, Asp, Gln, Glu 3-PhO CH═CH 12 59 Cys, Met, Ser, Thr 3-PhO CH═CH 13 nd Arg, Gly, His, Pro 3-PhO CH═CH 14 30 Lys(2-Cl-Cbz), 3-PhO CH═CH Phe, Trp, Tyr 15 48 Ala, Ile, Leu, Val 3-PhO CH═CH 16 nd Glu, Asp 2,3-benzo CH═CH 17 nd Cys, Met 2,3-benzo CH═CH 18 nd Ser, Thr 2,3-benzo CH═CH 19 nd His, Arg(Mtr) 2,3-benzo CH═CH 20 nd Pro, Gly 2,3-benzo CH═CH 21 nd Phe, Tyr 2,3-benzo CH═CH 22 nd Trp, Lys(2-Cl-Cbz) 2,3-benzo CH═CH 23 nd Ile, Ala 2,3-benzo CH═CH 24 nd Val, Leu 2,3-benzo CH═CH 25 nd Asn, Lys 2,3-benzo CH═CH 26 nd Ala, Ile 3,4-benzo CH═CH 27 nd Arg(Mtr), 3,4-benzo CH═CH Lys(2-Cl-Cbz) 28 nd Asp, Glu 3,4-benzo CH═CH 29 nd Cys, Met 3,4-benzo CH═CH 30 nd Gly, Pro 3,4-benzo CH═CH 31 nd His, Lys 3,4-benzo CH═CH 32 nd Leu, Val 3,4-benzo CH═CH 33 nd Lys(2-Cl-Cbz), Phe 3,4-benzo CH═CH 34 nd Ser,Thr 3,4-benzo CH═CH 35 nd Trp,Tyr 3,4-benzo CH═CH

TABLE 3

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R⁹ W,Q MS MH+ 36 nd CH₃ 4-CF₃ H CH═CH 458 37 19 H 4-CF₃ H CH═CH 430 38 nd (CH₂)₄NH(2-Cl-Cbz) 4-F H CH═CH 448 40 nd CH₃ 4-F H CH═CH 223 41 nd CH₂CO₂H 4-F H CH═CH 266 42 nd CH₂CH₂CO₂H 4-F H CH═CH 281 43 nd (CH₂)₃NHC(═NH)NH₂ 4-F H CH═CH 308 45 nd PhCH₂ 4-F H CH═CH 299 46 nd 4-HO-PhCH₂ 4-F H CH═CH 315 47 nd CH₂OH 4-F H CH═CH 238 48 nd CH(OH)CH₃ 4-F H CH═CH 253 49 1 (CH₂)₃NHC(═NH)NH₂ H H S 419 50 −6 (CH₂)₄NH₂ H H S 391 51 nd CH(CH₃)CH₂CH₃ H H S 376 52 21 CH₂CH₂CO₂H H H S 392 53 14 CH₂CO₂H H H S 378 54 18 CH₃ H H S 334 55 4 CH₂CH₂CONH₂ H H S 391 56 nd (CH₂)₄NHCbz H Me S 539 57 0 (CH₂)₄NHCbz H CH₂Ph S 615 58 nd CH₂(indol-3-yl) H Me S 463 59 26 CH₂CH₂CO₂-t-Bu H Me S 462 60 9 CH₂CH₂CO₂Et H Me S 434 61 14 CH₂CH₂CO₂H H Me S 406

TABLE 4

EPO/EBP-Ig cpd % inh @ 5 μM R^(a) R² R⁴ R⁹ MS, MH+ 62 nd t-Bu NO₂ NO₂ t-Bu 516 63 20 H PhCH₂NH PhO H 511 64 −4 H 4-MePhCONH 4-MePhCONH H 580 65 −7 H 4-MePhSO₂NH 4-MePhSO₂NH H 652 66 −16 H 3-ClPhCH₂NH PhO H 546 67 −8 H 3-BrPhCH₂NH PhO H 590 68 −13 H 2-FPhCH₂NH PhO H 529 69 −13 H 2-MePhCH₂NH PhO H 525 70 −8 H 4-FPhCH₂NH PhO H 529 71 −6 H 3-ClPhCH₂NH 4-Me-PhO H 560 72 −14 H F₅-PhCH₂NH 4-Me-PhO H 615 73 −13 H 2-FPhCH₂NH 4-Me-PhO H 543 74 −7 H 3-CNPhCH₂NH 4-Me-PhO H 550 75 −2 H PhCH₂NH 4-Me-PhO H 525

TABLE 5

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁹ n MH+ 76 25 t-Bu PhO H PhO H t-Bu 1 636 77 52 H PhO H PhO H H 1 524 78 nd H H 4-MePhCONH H BnO H 2 593 79 nd H H n-BuCONH H BnO H 2 559 80 nd H H 2-naphthyl CONH H BnO H 2 629 81 nd H H 2-furyl CONH H BnO H 2 569 82 32 H H 4-MeO-PhCONH H BnO H 2 609 83 18 H H HO₂C(CH₂)₃CONH H BnO H 2 589 84 14 H H C₂F₅CONH H BnO H 2 621 85 20 H H CF₃CONH H BnO H 2 571 86 37 H H 4-pyridyl-CONH H BnO H 2 580 87 23 H H 4-MePhSO₂NH H BnO H 2 629 88 10.3 H H HO₂CCH₂(1,1- H BnO H 2 643 cyclopentyl) CH₂CONH 89 22 H H PhOCONH H BnO H 2 595 90 29 H H 4-Ph-PhCONH H BnO H 2 655 91 19 H H 4-NO₂-PhCONH H BnO H 2 624

TABLE 6

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ N R⁹ MH+  92 20 H H H H H 2 Me 394  93 20 t-Bu H H H H 2 Me 450  94 25 Et H H H H 2 Me 422  95 15 t-Bu 2,3-benzo 2,3-benzo 2 Me 550  96 −5 t-Bu PhO H PhO H 2 Me 634  97 14 t-Bu 3,4-benzo 3,4-benzo 2 H 536  98 12 t-Bu H Ph H Ph 2 Me 602  99 13 t-Bu 3,4-di-Cl-PhO H 3,4-di-Cl-PhO H 2 Me 772 100 34 H H Ph H Ph 2 Me 546 101 32 H 3,4-di-Cl-PhO H 3,4-di-Cl-PhO H 2 Me 716 102  5 t-Bu 4-t-Bu-PhO H 4-t-Bu-PhO H 2 t-Bu 789 103 17 t-Bu 3-CF3-PhO H 3-CF3-PhO H 2 t-Bu 812 104 78 H 4-t-Bu-PhO H 4-t-Bu-PhO H 2 H 676 105 70 H 3-CF3-PhO H 3-CF3-PhO H 2 H 700 106 20 t-Bu PhO H PhO H 1 t-Bu 662 107 78 H PhO H PhO H 2 H  562* 108 81 H PhO H PhO H 1 H 550 *[M-H]⁻

TABLE 7

EPO/EBP-Ig cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁹ MS, MH+ 109  7 Boc BnO H BnO H Me 653 110 54 H H BnO H BnO Me 553 111  5 Boc H BnO H BnO Me 653 112 59 H BnO H BnO H Me 553 113 24 Boc H BnO NO₂ H Me 592 114 37 H H BnO NO₂ H Me 492 115 35 H H BnO NH₂ H Me 462 116 32 H H BnO n-BuCONH H Me 546 117 34 H H BnO 2-furylCONH H Me 556 118 36 H H BnO 4-MePhCONH H Me 580 119 34 H H BnO i-Pr-CONH H Me 532 120 35 H H BnO 4-pyridyl- H Me 567 CONH 121 45 H H BnO 2-naphthyl- H Me 616 CONH 122 nd Boc PhCH₂NH H PhCH₂NH H Me 651 123 nd Boc 2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 124 nd Boc 4-MeO- H 4-MeO- H Me 711 PhCH₂NH PhCH₂NH 125 nd Boc 3,4-di-MeO- H 3,4 di-MeO- H Me 771 PhCH₂NH PhCH₂NH 126 nd Boc —NH₂ H —NH₂ H Me 471 127 nd H PhCH₂NH H PhCH₂NH H Me 551 128 nd H 2-MePhCH₂NH H 2-MePhCH₂NH H Me 579 129 nd H 4-MeO- H 4-MeO- H Me 611 PhCH₂NH PhCH₂NH 130 nd H 3,4-di-MeO- H 3,4-di-MeO- H Me 671 PhCH₂NH PhCH₂NH 131 nd H PhCH₂CH₂NH H PhCH₂CH₂NH H Me 579 132 nd HO₂CCH₂ PhCH₂NH H PhCH₂NH H Me 651 CH₂CO 133 nd HO₂CCH₂ 2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 CH₂CO 134 nd HO₂CCH₂ 4-MeO- H 4-MeO- H Me 711 CH₂CO PhCH₂NH PhCH₂NH 135 nd HO₂CCH₂ 3,4-di-MeO- H 3,4-di-MeO- H Me 771 CH₂CO PhCH₂NH PhCH₂NH 136 nd HO₂CCH₂ PhCH₂CH₂NH H PhCH₂CH₂NH H Me 679 CH₂CO

TABLE 8

EPO/EBP-Ig cpd % inh @ 50 μM R^(a) R² R⁴ R⁵ R⁹ MS, MH+ 137 nd H PhO PhO H Me 551 138 nd Boc 4-t-Bu-PhO BnO H Me 721 139 nd H 4-t-Bu-PhO BnO H Me 621 140 nd H (CF₃CO)₂N BnO H H 666 141 nd H PhCONH BnO H H 578 142 nd H 4-pyridyl-CONH BnO H H 579 143 nd H (CF₃CO)₂N PhO H H 652 144 nd H PhCONH PhO H H 564 145 nd H 4-pyridyl-CONH PhO H H 565 146 nd H (CF₃CO)₂N MeO MeO H 620 147 nd H PhCONH MeO MeO H 532 148 nd H 4-pyridyl-CONH MeO MeO H 533 149 nd H (CF₃CO)₂N H PhO H 652 150 nd H PhCONH H PhO H 564 151 nd H 4-pyridyl-CONH H PhO H 565 152 nd H PhCONH H BnO H 578 153 nd H 4-pyridyl-CONH H BnO H 579 154 nd H (CF₃CO)₂N H BnO H 666 155 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH PhO H H 694 156 nd HO₂CCH₂CH₂CO PhCONH PhO H H 664 157 nd HO₂CCH₂CH₂CO 2-naphthyl-CONH PhO H H 714 158 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH PhO H H 714 159 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH 2,3-benzo H 652 160 nd HO₂CCH₂CH₂CO PhCONH 2,3-benzo H 622 161 nd HO₂CCH₂CH₂CO 2-naphthyl-CONH 2,3-benzo H 672 162 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH 2,3-benzo H 672 163 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH H F H 620 164 nd HO₂CCH₂CH₂CO PhCONH H F H 590 165 nd HO₂CCH₂CH₂CO 2-naphthyl-CONH H F H 640 166 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH H F H 640 167 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH BnO H H 708 168 nd HO₂CCH₂CH₂CO PhCONH BnO H H 678 169 nd HO₂CCH₂CH₂CO 2-naphthyl-CONH BnO H H 728 170 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH BnO H H 728

TABLE 9

EPO/EBP-Ig cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁹ MS, MH+ 171 nb Cbz H H H H Me 527 172 15 Cbz H H H H H 513 173  5 Cbz H H H H t-Bu 569 174 23 Cbz H MeO H MeO Me 587 175  1 Cbz 3,4-benzo 3,4-benzo Me 627 176 −4 Cbz PhO H PhO H Me 711 177 nd Cbz 2,3-benzo 2,3-benzo Me 627 178 36 Boc H NO₂ H NO₂ Me 583 179 30 Boc H NO₂ H NO₂ H 569 180 −4 Boc PhO H PhO H Me 677 181 −9 Boc 4-t-Bu-PhO H 4-t-Bu-PhO H Me 790 182 18 H 4-t-Bu-PhO H 4-t-Bu-PhO H Me 689 183 36 Boc NO₂ H NO₂ H Me 583 184 53 H NO₂ H NO₂ H Me 483 185 29 H NH₂ H NH₂ H Me 423 186 nd H n-Bu-CONH H n-Bu-CONH H Me 591 187 nd H 2-furyl-CONH H 2-furyl-CONH H Me 611 188 nd H PhCONH H PhCONH H Me 631 189 nd H 4-Me-PhCONH H 4-Me-PhCONH H Me 659 190 nd H 4-NO₂-PhCONH H 4-NO ₂-CONH H Me 721 191 nd H 4-Me-PhSO₂NH H 4-Me-PhSO₂CONH H Me 731 192 nd H Cbz-NH H Cbz-NH H Me 691 193 nd H 4-Br-PhCONH H 4-Br-PhCONH H Me 789 194 nd H 2-MeO-PhCONH H 2-MeO-PhCONH H Me 691 195 nd H 3-MeO-PhCONH H 3-MeO-PhCONH H Me 691 196 nd H 4-MeO-PhCONH H 4-MeO-PhCONH H Me 691 197 nd H CH₃CH═CHCONH H CH₃CH═CHCONH H Me 559 198 nd H C₂F₅CONH H C₂F₅CONH H Me 715 199 nd H 2-naphthyl-CONH H 2-naphthyl-CONH H Me 731 200 nd H EtO₂CCH₂CH₂CONH H EtO₂CCH₂CH₂CONH H Me 679 201 nd H CF₃CONH H CF₃CONH H Me 615 202 nd H MeSO₂NH H MeSO₂NH H Me 579

TABLE 10

EPO/EBP-Ig cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ Z MS, MH+ 203 37 Boc H H H H 4-(MeCOCH₂CH₂)-PhNH 640 204 −6 H H H H H 4-(MeCOCH₂CH₂)-PhNH 540 205 26 H H H H H n-Bu-NH 434 206 17 2-MeO-PhCO H H H H n-Bu-NH 568 207 20 4-MeO-PhCO H H H H n-Bu-NH 568 208 22 PhCO H H H H n-Bu-NH 538 209 25 2-MeO-PhCO H H H H n-Bu-NH 568 210 nd Boc H H H H 4-MeO-PhCH₂CH₂NH 612 211 62 H H H H H 4-MeO-PhCH₂CH₂NH 512 212 −10  H H H H H n-Pr-NH 420 214 nd Boc H H H H 3,4-di-MeO- 642 PhCH₂CH₂NH 215 nd Boc H H H H 3-MeO-PhCH₂CH₂NH 612 216 10 Boc H H H H 4-(PhCH═CHCH₂O)- 700 PhCH₂NH 217 nd Boc H H H H 4-HO-PhCH₂NH 584 218 nd Boc H H H H EtNH 506 219 nd Boc H H H H MeNH 492 220 45 H H H H H 4-(PhCH═CHCH₂O)- 600 PhCH₂NH 221 48 H H H H H 3,4-di-MeO- 542 PhCH₂CH₂NH 222 56 H H H H H 3-MeO-PhCH₂CH₂NH 512 223 nd Boc H H H H 2-MeO-PhCH₂CH₂NH 612 224 51 H H H H H 2-MeO-PhCH₂CH₂NH 512 225 10 Boc PhO H PhO H 4-MeO-PhCH₂CH₂NH 797 226 nd Boc H H H H PhCH₂CH₂NH 582 227 48 H H H H H PhCH₂CH₂NH 482 228 21 PhNHCO PhO H PhO H 4-MeO-PhCH₂CH₂NH 816 229 22 4-PhO-PhNHCO H H H H 4-MeO-PhCH₂CH₂NH 723 230 42 3,4-di-Cl-PhNHCO H H H H 4-MeO-PhCH₂CH₂NH 700 231 36 4-EtO2C-PhNHCO H H H H 4-MeO-PhCH₂CH₂NH 703 232 14 4-PhO-PhNHCO PhO H PhO H 4-MeO-PhCH₂CH₂NH 908 233 18 H H NO₂ H NO₂ 3-MeO-PhCH₂CH₂NH 602 234 nd Boc H H H H PhCH₂NH 568 235 49 H H H H H PhCH₂NH 468 236 nd Boc H Ph H Ph 4-MeO-PhCH₂CH₂NH 765 237 55 HO₂CCH₂CH₂CO H H H H 3-MeO-PhCH₂CH₂NH 612 238 39 H H Ph H Ph 4-MeO-PhCH₂CH₂NH 664 239 46 H PhO H PhO H PhCH₂CH₂NH 666 240 nd HO₂CCH₂CH₂CH₂CO PhO H PhO H PhCH₂CH₂NH 780 285 40 H H H H H 4-(NH₂CO)piperidin-1-yl 489

TABLE 11

EPO/ EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R³ R⁴ R⁵ Z r [MH₂/2]+ 241 2 t-Bu H BnO H BnO NH(CH₂)₃O(CH₂)₄O 1 666 (CH₂)₃NH 242 1 t-Bu H BnO H BnO NH(CH₂)₃O(CH₂CH 1 674 ₂O)₂(CH₂)₃NH 243 75 H H BnO H BnO NH(CH₂)₃O(CH₂)₄O 1 610 (CH₂)₃NH 244 66 H H BnO H BnO NH(CH₂)₃O(CH₂CH 1 618 ₂O)₂(CH₂)₃NH 245 0 t-Bu H BnO H BnO NH(CH₂)₂O(CH₂)₂O 2 652 (CH₂)₂NH 246 79 H H BnO H BnO NH(CH₂)₂O(CH₂)₂O 2 596 (CH₂)₂NH 247 47 H n-Bu- H PhO H NH(CH₂)₂O(CH₂)₂O 2 575 CONH CH₂)₂NH 248 56 H 2-furyl- H PhO H NH(CH₂)₂O(CH₂)₂O 2 585 CONH (CH₂)₂NH 249 72 H 4-Me- H PhO H NH(CH₂)₂O(CH₂)₂O 2 609 PhCONH (CH₂)₂NH 250 78 H 4-Me- H PhO H NH(CH₂)₂O(CH₂)₂O 2 645 PhSO₂NH (CH₂)₂NH

TABLE 12

EPO/EBP-Ig MS cpd % inh @ 50 μM R^(a) R² R⁴ Z r [MH₂/2]+ 251 49 H H H NH(CH₂)₂O(CH₂)₂ 2 436 O(CH₂)₂NH 252 −4 t-Bu 4-t-Bu-PhO 4-t-Bu-PhO NH(CH₂)₃O(CH₂)₄ 1 803 O(CH₂)₃NH 253 −5 t-Bu 4-t-Bu-PhO 4-t-BuPhO NH(CH₂)₃O(CH₂C 1 811 H₂O)₂(CH₂)₃NH 254 −9 t-Bu 4-t-Bu-PhO 4-t-Bu-PhO NH(CH₂)₁₀NH 1 787 255 0 t-Bu 4-t-Bu-PhO 4-t-Bu-PhO NH(CH₂)₁₂NH 1 801 256 10 t-Bu 4-t-Bu-PhO 4-t-Bu-PhO NH(CH₂)₂O(CH₂)₂ 1 789 O(CH₂)₂NH

TABLE 13

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) Z [MH₂/2]+ 257 −26 Boc NH(CH₂)₃O(CH₂CH₂O)₂ 731 (CH₂)₃NH 258 −24 Boc NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 723 259 −13 Boc NH(CH₂)₁₂NH 721 260 −12 Boc NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 695 261 51 H NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 595 262 93 HO₂CCH₂CH₂CO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 695 263 88 HO₂C(CH₂)₃CO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 709 264 89 HO₂CCH₂CMe₂CH₂CO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 737 265 65 HO₂CCH₂CH₂CO NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 723 266 82 HO₂C(CH₂)₃CO NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 737 267 83 HO₂CCH₂CMe₂CH₂CO NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 765 268 40 HO₂CCH₂CMe₂CH₂CO NH(CH₂)₁₂NH 764 269 55 HO₂CCH₂CH₂CH₂CO NH(CH₂)₁₂NH 735 270 56 HO₂CCH₂CH₂CO NH(CH₂)₁₂NH 721 271 77 HO₂CCH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂ 731 (CH₂)₃NH 272 78 HO₂CCH₂CH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂ 745 (CH₂)₃NH

TABLE 14

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R⁴ Z n [MH₂/2]+ 273 nd HO₂CCH₂C 4-Me-PhO 4-Me-PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 695 H₂CO 274 nd HO₂CCH₂C PhO PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 667 H₂CO 275 nd HO₂CCH₂C 4-MeO-PhO 4-MeO-PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 727 H₂CO 276 nd HO₂CCH₂C 4-t-Bu-PhO 4-t-Bu-PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 780 H₂CO 277 nd H PhO PhO (NHCH₂CH₂)₃N 3 813 278 nd H 4-Me-PhO 4-Me-PhO (NHCH₂CH₂)₃N 3 855 279 nd H 4-MeO-PhO 4-MeO-PhO (NHCH₂CH₂)₃N 3 903 280 nd HO₂CCH₂C 4-MeO-PhO 4-MeO-PhO (NHCH₂CH₂)₃N 3 1053  H₂CO 281 nd HO₂CCH₂C 4-Me-PhO 4-Me-PhO (NHCH₂CH₂)₃N 3 1005  H₂CO 282 nd HO₂CCH₂C PhO PhO (NHCH₂CH₂)₃N 3 963 H₂CO 283 nd Boc PhO PhO NH(CH₂)₃NMe 2 666 (CH₂)₃NH 284 nd Boc 4-Me-PhO 4-Me-PhO NH(CH₂)₃NMe 2 694 (CH₂)₃NH

TABLE 15

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ MS, MH+ 285 −28   Me H H 473 286 46 H BnO H 565 287 36 H 4-Me-PhO H 565 288 27 H 4-tBu-PhO H 607 289 20 H H PhO 551

TABLE 16A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 290  0 Me PhO H t-Bu 291 17 Me H Ph t-Bu 292 11 Me 3-CF3-C6H4O H t-Bu 293 14 Me 3,4-Cl2-C6H3O H t-Bu 294  9 Me 4-t-Bu-C6H4O H t-Bu 295 10 Me H Ph H 296  0 Me 3,4-Cl2-C6H3O H H 297  0 Me 3-CF3-C6H4O H H 298  1 Me 4-t-Bu-C6H4O H H 299 nd Me PhO H H 300 57 H PhO H H 301 25 H H Ph t-Bu 302 30 H 3,4-Cl2-C6H3O H t-Bu 303 21 H 3-CF3-C6H4O H t-Bu 304 19 H 4-t-Bu-C6H4O H t-Bu 305 48 H H Ph H 306 21 Me H H t-Bu 307 25 H 3,4-Cl2-C6H3O H H 308 25 H 3-CF3-C6H4O H H 309 13 H 4-t-Bu-C6H4O H H 310 34 Me H H H

TABLE 16B MS, cpd MPLC solvent appearance empirical formula MH+ 290 10-30% pale yellow oil C38H41NO5 592 EtOAc/hex 291 1:5 EtOAc/hex yellow oil C38H41NO3 560 292 1:5 EtOAc/hex yellow oil C40H39F6NO5 728 293 1:5 EtOAc/hex yellow oil C38H37C14NO5 728 294 1:5 EtOAc/hex yellow oil C46H57NO5 704 295 off-white solid C34H33NO3/1 504 C2H4O2 296 amber solid C34H29C14NO5/1 672 C2H4O2 297 amber oil C36H31F6NO5/1 672 C2H4O2 298 off-white solid C42H49NO5/1 648 C2H4O2 299 30% EtOAc/hex colorless oil C34H33NO5 536 300 sticky C33H31NO5/1 522 yellow solid C2H4O2 301 yellow solid C37H39NO3 546 302 amber oil C37H35C14NO5 714 303 amber oil C39H37F6NO5 714 304 amber oil C45H55NO5 690 305 amber solid C33H31NO2/1 490 C2HF3O2 306 light-yellow oil C26H33NO3/0.25 408 H2O 307 amber solid C33H27C14NO5/1 658 C2HF3O2 308 amber oil C35H29F6NO5/1 658 C2HF3O2 309 off-white solid C41H47NO5/1 634 C2HF3O2 310 light yellow oil C22H25NO3/1 352 C2H4O2

TABLE 17A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ 311 5.3 t-Bu PhO H 312 45 H PhO H

TABLE 17B cpd MPLC solvent appearance empirical formula MS, MH+ 311 10% EtOAc/hex pale yellow oil C36H37NO4 548 312 sticky brown C32H29NO4/1 492 solid C2HF3O2

TABLE 18A

EPO/EBP-Ig, cpd % inh @ 50 μM R¹ R² R³ R⁴ 313 28 H H CF3 (CH2)4NH(2Cl-Cbz) Cbz 314 12 Me H CO2H (CH2)4NH2 315 nd Me H NO2 (CH2)4NHBoc 316 20 Me OPh H (CH2)4NHBoc 317 13 Me 4-t-Bu-C6H4O H (CH2)4NHBoc 318 14 Me H H (CH2)4NHCbz 319 nd Me H H (CH2)4NHCbz 320 17 Me H OMe (CH2)4NHCbz 321 42 Me CO2Me H (CH2)4NHCbz 322 nd Me H 2,3-benzo (CH2)4NHCbz 323  6 Me H CO2H (CH2)4NHCbz 324 nd Me H CO2Me nd

TABLE 18B MS, cpd MPLC solvent appearance empirical formula MH+ 313 yellow oil C25H28ClF3N2O4\1 499 C2HF3O2 314 yellow oil C17H24N2O4 321 315 30% EtOAc/hex dark yellow C21H31N3O6 422 gum 316 20-50% pale yellow oil C27H36N2O5 469 EtOAc/hex 317 pale yellow oil C31H44N2O5 525 318 gum C24H30N2O4 411 319 pale yellow oil C24H30N2O4 411 320 2% MeOH/ yellow oil C25H32N2O5 441 CH2Cl2 321 yellow oil C26H32N2O6\1 469 C2H4O2 322 25-50% clear residue C28H32N2O4 461 EtOAc/hex 323 yellow oil C25H30N2O6 455 324 yellow oil C26H32N2O6 469

TABLE 19A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 325  6 Me OPh H CH2CH2CO2H 326  0 H OPh H CH2CH2CO2H 327 11 Me H Ph CH2CH2CO2H 328 33 Me 3,4-Cl2-C6H3O H CH2CH2CO2H 329 13 H H Ph CH2CH2CO2H 330 12 H 3-CF3-C6H4O H CH2CH2CO2H 331 18 H 4-t-Bu-C6H4O H CH2CH2CO2H 332 17 H 3,4-Cl2-C6H3O H CH2CH2CO2H 333 16 Me 3,4-benzo CH2CH2CO2-t-Bu 334  6 Me OPh H CH2CH2CO2-t-Bu 335 25 Me H Ph CH2CH2CO2-t-Bu 336 32 Me 3,4-Cl2-C6H3O H CH2CH2CO2-t-Bu 337  0 H OPh H CH2CH2CO2-t-Bu 338 23 t-Bu 3-CF3-C6H4O H CH2CH2CO2-t-Bu 339 10 t-Bu 4-t-Bu-C6H4O H CH2CH2CO2-t-Bu 340 14 H H Ph CH2CH2CO2-t-Bu 341 19 H 3,4-Cl2-C6H3O H CH2CH2CO2-t-Bu

TABLE 19B MS, cpd MPLC solvent appearance empirical formula MH+ 325 off-white solid C21H23NO5\1 370 C2F3HO2 326 fluffy white C20H21NO5\1 356 solid C2HF3O2 327 off-white solid C21H23NO4\1 354 C2F3HO2 328 amber oil C21H21C12NO5\1 438 C2F3HO2 329 amber solid C20H21NO4\1 340 C2HF3O2 330 amber oil C21H20F3NO5\1 424 C2HF3O2 331 amber oil C24H29NO5\1 412 C2HF3O2 332 amber oil C20H19CL2NO5\1 424 C2HF3O2 333 10-25% EtOAc/hex yellow oil C23H29NO4 384 334 10-30% EtOAc/hex pale yellow oil C25H31NO5 426 335 1:5 EtOAc/hex yellow oil C25H31NO4 410 336 1:5 EtOAc/hex yellow oil C25H29C12NO5 494 337 white powder C24H29NO5\0.4 412 H2O 338 1:5 EtOAc/hex yellow oil C29H36F3NO5 536 339 1:5 EtOAc/hex yellow oil C32H45NO5 524 340 yellow solid C24H29NO4 396 341 white solid C24H27C12NO5 480

TABLE 20A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 342  0 Me H Ph CH2OH 343 37 Me 4-t-Bu-C6H4O H CH2OH 344  4 Me 3-CF3-C6H4O H CH2OH 345 40 Me 3,4-Cl2-C6H3O H CH2OH 346 28 Me OPh H CH2OH 347 23 H OPh H CH2OH 348 21 H H Ph CH2OH 349 23 H 3,4-Cl2-C6H3O H CH2OH 350 23 H 3-CF3-C6H4O H CH2OH 351 29 H 4-t-Bu-C6H4O H CH2OH 352  8 Me OPh H CH2O-t-Bu 353 24 Me H Ph CH2O-t-Bu 354 31 Me 3,4-Cl2-C6H3O H CH2O-t-Bu 355 22 Me 3-CF3-C6H4O H CH2O-t-Bu 356 23 Me 4-t-Bu-C6H4O H CH2O-t-Bu 357 12 H 3-CF3-C6H4O H CH2O-t-Bu

TABLE 20B MS, cpd MPLC solvent appearance empirical formula MH+ 342 off-white solid C19H21NO3\1 312 C2F3HO2 343 amber oil C23H29NO4\1 384 C2F3HO2 344 amber oil C20H20F3NO4\1 396 C2F3HO2 345 amber oil C19H19C12NO4\1 396 C2F3HO2 346 EtOAc pale yellow oil C19H21NO4 328 347 amber oil C18H19NO4\1 314 C2HF3O2 348 yellow solid C18H19NO3\1 298 C2HF3O2 349 amber oil C18H17C12NO4\1 382 C2HF3O2 350 amber oil C19H18F3NO4\1 382 C2HF3O2 351 amber oil C22H27NO4\1 370 C2HF3O2 352 10-30% EtOAc/hex pale yellow oil C23H29NO4 384 353 20% EtOAc/hex off-white solid C23H29NO3 368 354 20% EtOAc/hex yellow oil C23H27C12NO4 452 355 20% EtOAc/hex yellow oil C24H28F3NO4 452 356 20% EtOAc/hex yellow oil C27H37NO4 440 357 white solid C23H26F3NO4 438

TABLE 21A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 358  0 H H CF3 (s)-CH(OH)CH3 359 25 Me CO2Me H (s)-CH(OMe)CH3 360 18 Me H H Bn 361 24 Me CO2Me H Bn 362  0 H H CF3 CH2(4-HOC6H4) 363 33 Me CO2Me H CH2(4-MeOC6H4) 364 16 Me H H CH2(indol-3-yl) 365  0 H H CF3 CH2CH2SMe 366 38 Me CO2Me H CH2CO2Me 367  0 H H CF3 CH2CONH2 368 40 Me CO2Me H CH2SBn 369 12 H H CF3 i-Bu 370  0 H H CF3 i-Pr 371 16 Me CO2Me H i-Pr 372  0 Me H H Me

TABLE 21B MS, cpd MPLC solvent appearance empirical formula MH+ 358 amber oil C14H16F3NO3\1 304 C2HF3O2 359 amber oil C17H23NO5\1 322 C2H4O2 360 20% EtOAc/hex light-yellow oil C19H21NO2 296 361 amber oil C22H25NO5\1 354 C2H4O2 362 amber oil C19H18F3NO3\1 366 C2HF3O2 363 amber oil C22H25NO5\1 384 C2H4O2 364 1:2 EtOAc/hex tan solid C21H22N2O2 335 365 amber oil C15H18F3NO2S\1 334 C2HF3O2 366 amber oil C17H21NO6\1 336 C2H4O2 367 amber oil C14H15F3N2O3\1 317 C2HF3O2 368 amber oil C22H25NO4S\1 400 C2H4O2 369 amber oil C17H22F3NO2\1 316 C2HF3O2 370 amber oil C15H18F3NO2\1 302 C2HF3O2 371 amber oil C17H23NO4\1 306 C2H4O2 372 20% EtOAc/hex yellow oil C13H17NO2\0.10 220 C4H8O2 

What is claimed is:
 1. A compound of the formula below:

wherein R² and R⁴ are both 4-t-Bu-PhO; R³, R⁵, R⁹, and R^(a) are all H; and n is 1 or
 2. 2. A pharmaceutical composition comprising an active drug component and a compound of the formula

wherein R² and R⁴ are both 4-t-Bu-PhO; R³, R⁵, R⁹, and R^(a) are all H; and n is 1 or
 2. 3. An oral composition comprising an EPO receptor modulating compound of the formula

wherein R² and R⁴ are both 4-t-Bu-PhO; R³, R⁵, R⁹, and R^(a) are all H; and n is 1 or 2; said composition having an active drug component being combined with an oral, non-toxic pharmaceutically acceptable inert carrier.
 4. A pharmaceutical composition comprising the compound of claim
 1. 5. The composition of claim 2 wherein said active drug component is combined with an oral, non-toxic pharmaceutically acceptable inert carrier.
 6. The composition of claim 5 wherein said carrier is ethanol, glycerol, or water.
 7. The composition of claim 2 further comprising binders, lubricants, disintegrating agents or coloring agents.
 8. The composition of claim 7 wherein said binders are selected from the group consisting of starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums carboxymethylcellulose, polyethylene glycol, and waxes.
 9. The composition of claim 7 wherein said lubricants are selected from the group consisting of sodium oleate, sodium iterate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.
 10. The composition of claim 7 wherein said disintegrating agents are selected from the group consisting of starch, methyl cellulose, agar, bentonite, and xanthan gum.
 11. The composition of claim 2 contained in a topical administration.
 12. The Composition of claim 11 wherein said active drug component can be admixed with carrier materials selected from the group consisting of alcohols, aloe vera gel, allontoin, glycerine, vitamin A and E oils, mineral oil, and PPG2 myristyl propionate.
 13. The composition of claim 3 wherein said carrier is ethanol, glycerol, or water.
 14. The composition of claim 3 further comprising binders, lubricants, disintegrating agents or coloring agents.
 15. The composition of claim 14 wherein said binders are selected from the group consisting of starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums carboxymethylcellulose, polyethylene glycol, and waxes.
 16. The composition of claim 14 wherein said lubricants are selected from the group consisting of sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.
 17. The composition of claim 14 wherein said disintegrating agents are selected from the group consisting of starch, methyl cellulose, agar, bentonite, and xanthan gum. 